neural architecture of the primary gustatory center of ... · phageal ganglion (sog) of the brain....

Neural Architecture of the Primary Gustatory Centerof Drosophila melanogaster Visualized With GAL4 andLexA Enhancer-Trap Systems

Takaaki Miyazaki1,2 and Kei Ito1,2*1Institute of Molecular and Cellular Biosciences (IMCB), The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan2Department of Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa,

Chiba 277-0882, Japan

ABSTRACTGustatory information is essential for animals to select

edible foods and avoid poisons. Whereas mammals

detect tastants with their taste receptor cells, which

convey gustatory signals to the brain indirectly via the

taste sensory neurons, insect gustatory receptor neu-

rons (GRNs) send their axons directly to the primary

gustatory center in the suboesophageal ganglion (SOG).

In spite of this relatively simple architecture, the pre-

cise structure of the insect primary gustatory center

has not been revealed in enough detail. To obtain com-

prehensive anatomical knowledge about this brain area,

we screened the Drosophila melanogaster GAL4

enhancer-trap strains that visualize specific subsets of

the gustatory neurons as well as putative mechanosen-

sory neurons associated with the taste pegs. Terminals

of these neurons form three branches in the SOG. To

map the positions of their arborization areas precisely,

we screened newly established LexA::VP16 enhancer-

trap strains and obtained a driver line that labels a

large subset of peripheral sensory neurons. By double-

labeling specific and landmark neurons with GAL4 and

LexA strains, we were able to distinguish 11 zones in

the primary gustatory center, among which 5 zones

were identified newly in this study. Arborization areas

of various known GRNs on the labellum, oesophagus,

and legs were also mapped in this framework. The pu-

tative mechanosensory neurons terminate exclusively in

three zones of these areas, supporting the notion of

segregated primary centers that are specialized for che-

mosensory and mechanosensory signals associated

with gustatory sensation. J. Comp. Neurol. 518:4147–

4181, 2010.

VC 2010 Wiley-Liss, Inc.

INDEXING TERMS: Drosophila; insect; gustatory receptor neuron; brain; suboesophageal ganglion; enhancer trap; GAL4;

LexA

Gustatory information is an important sensory modality

for animals to select edible foods and avoid poisons.

Mammals detect tastants with their taste receptor cells

(TRCs) in the taste buds of the oral cavity (Fig. 1A). TRCs

transmit gustatory signals to the taste neurons beneath

the taste buds, which project through the facial (VII), glos-

sopharyngeal (IX), and vagus (X) nerves to terminate in

the nucleus of the solitary tract (NST). From there signals

are further conveyed to the thalamus and higher areas of

the brain (Buck, 2000).

Insects also use gustatory information for finding

foods. In addition, taste stimuli play important roles in

communications among individuals. The proper mating

behavior sequence of the German cockroach Blattella

germanica requires nonvolatile cues secreted from the

tergal glands (Nojima et al., 1999b), which also function

as a general dietary feeding stimulant (Nojima et al.,

1999a,b; Kugimiya et al., 2002; Nojima et al., 2002). The

gustatory system is also involved in regulation of court-

ship rituals in male Drosophila (Miyamoto and Amrein,

2008; Koganezawa et al., 2010). Social insects like ter-

mites and ants use the taste of hydrocarbon in their body

wax to distinguish their own colony members from aliens

Additional Supporting Information may be found in the online version ofthis article.

Grant sponsor: Human Frontier Science Program; Grant sponsor:Ministry of Education, Culture, Sports, Science and Technology of Japan(Grant-in-Aid for Scientific Research to K.I.); Grant sponsor: Japan Societyfor the Promotion of Science (predoctoral fellowship to T.M.).

*CORRESPONDENCE TO: Kei Ito, Institute of Molecular and CellularBiosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032,Japan. E-mail: [email protected]

VC 2010 Wiley-Liss, Inc.

Received April 13, 2009; Revised March 9, 2010; Accepted April 26, 2010

DOI 10.1002/cne.22433

Published online May 20, 2010 in Wiley Online Library (wileyonlinelibrary.com)

The Journal of Comparative Neurology | Research in Systems Neuroscience 518:4147–4181 (2010) 4147

RESEARCH ARTICLE

(Howard et al., 1978, 1982; Vander Meer et al., 1998;

Ozaki et al., 2005). In addition, taste sensation is involved

in the regulation of acquired behavior: associative learn-

ing of crickets, honeybees, and flies occurs between ol-

factory or visual sensation as conditioned stimuli and

taste as unconditioned stimuli (Hammer, 1993; Matsu-

moto and Mizunami, 2002; Farooqui et al., 2003;

Schwaerzel et al., 2003; Unoki et al., 2005,2006).

Insects have their main taste receptors on the surface

of the labellum of their mouthparts (Falk et al., 1976;

Figure 1

Miyazaki and Ito

4148 The Journal of Comparative Neurology |Research in Systems Neuroscience

Singh, 1997; Fig. 1B,C). In addition, they have taste sen-

sors along the oesophagus and on the tips of their legs

(Singh, 1997), and females of many insect species have

gustatory sensors on their ovipositors, with which they

can look for foods for their offspring when they lay eggs

(Rice, 1977; Taylor, 1989; Yang et al., 2008). Insect gus-

tatory sensors on the body surface are called sensilla or

pegs, because their shape is that of tiny bristles (Fig. 1D)

or pins (Fig. 1E), respectively. Tastants enter the sensil-

lum or peg through a pore on its tip and are dissolved in

the hemolymph in its lumen. Each sensillum is equipped

with one or a few gustatory receptor neurons (GRNs) and

one mechanosensory receptor neuron at its root (Fig.

1G). A peg is associated with one GRN and one mechano-

sensory neuron (Falk et al., 1976; Nayak and Singh,

1983; Singh, 1997).

The GRNs are bipolar, extending their dendrites into

the lumen of the sensilla/pegs and their axons to the cen-

tral nervous system (Fig. 1G). The axons deriving from the

labellar GRNs project via the labial nerve and terminate in

the primary gustatory center (PGC), which is housed in

the suboesophageal ganglion (SOG; Fig. 1C,H–J). The

GRNs along the oesophagus send their axons via the pha-

ryngeal nerve and terminate in the anterior area of the

SOG (Fig. 1C).

Whereas some insect species also have gustatory sen-

silla in the antennae and maxillary palps, those of the

fruitfly Drosophila melanogaster (Fig. 1F) have only olfac-

tory and mechanosensory sensilla (Stocker, 1994; Shanb-

hag et al., 1999). Unlike gustatory sensilla, olfactory sen-

silla have no associated mechanosensory neurons

(Stocker, 1994).

How does the gustatory sensory system discriminate

different tastes? In mammalian TRCs, different sets of G-

protein-coupled transmembrane molecules serve as gus-

tatory receptors: T2Rs, T1R2/T1R3 hetero-oligomers,

and T1R1/T1R3 hetero-oligomers for bitterness, sweet-

ness, and umami, respectively (Adler et al., 2000; Chan-

drashekar et al., 2000; Nelson et al., 2001, 2002; Zhao

et al., 2003; Mueller et al., 2005). A member of the group

of transient receptor potential channels (PKD2L1) and an

epithelial sodium channel are receptors for sourness and

Figure 1. Human and fly gustatory systems. A: Human gustatory neural pathways. Schematic sagittal section of a human head. Taste re-

ceptor cells (TRCs) are housed in the taste buds, which are distributed on the tongue and pharynx. They form synapses on the dendrites

of the taste sensory neurons. TRCs on the anterior two-thirds of the tongue, the posterior one-third of the tongue, and the pharynx are in-

nervated by the sensory neurons in the geniculate ganglion via the facial (VII) nerve, the petrosal ganglion via the glossopharyngeal (IX)

nerve, and the nodose ganglion via the vagal (X) nerve, respectively. These sensory neurons send gustatory information to the nucleus of

the solitary tract (NST) in the medulla oblongata, from where information is transmitted to higher order centers such as the thalamus and

gustatory cortex. B: Lateral view of a fly head (dorsal up, anterior to the left). The labellum is located in the tip of the proboscis. The max-

illary palp protrudes from the root of the proboscis. C: Schematic sagittal section of a fly head (dorsal up, anterior to the left). Unlike

TRCs in mammals, gustatory receptor neurons (GRNs) in the labellum send their axons directly through the labial nerve to the suboeso-

phageal ganglion (SOG) of the brain. Olfactory receptor neurons in the maxillary palp project through the maxillary nerve, which is merged

with the labial nerve before it enters the SOG. Gustatory neurons along the oesophagus project via the pharyngeal and accessory pharyn-

geal nerves, which enter a more anterior area of the SOG than the labial nerve. D,E: Lateral (D) and medial (E) views of the labellum,

which was cut at the midline. Three-dimensional (3D) reconstruction of the cuticular autofluorescence recorded with a confocal laser

microscope (dorsal up, anterior to the left). About 30 taste sensilla (white arrowheads) and two mechanosensory bristles (yellow arrows)

reside on the lateral surface of the labellum (D). The dotted rectangle indicates the area shown in Figures 2 and 7A–C. The medial recess

(E) features six groove structures called pseudotracheae (white arrows). About five taste pegs (white arrowheads) reside in front of each

pseudotrachea. F: Maxillary palp. Lateral view. Same preparations as in D (dorsal up, anterior to the left). G: Schematic diagram of a taste

sensillum. A sensillum has a pore on its tip, from where chemical compounds enter the internal lumen. It is equipped with two or four

GRNs and one mechanosensory neuron. The GRNs extend their dendrites into the lumen of the sensillum, whereas the dendrite of the

mechanosensory neuron ends at the root of the sensillum. Support cells surround the GRNs and mechanosensory neurons. H,I: Overall

structure of the fly brain. 3D reconstruction of the frontal view (H; dorsal up) and sagittal section (I; dorsal up, anterior to the left). In H,

the axons of the olfactory receptor neurons labeled with Or83b-GAL4 are visualized with UAS-DsRed reporter and anti-DsRed antibody (ma-

genta), whereas the axons labeled with LexAV-NV4 are visualized with lexAop-rCD2::GFP reporter (green) without immunolabeling. In I, the

axons labeled with the GAL4-LB6 strain are visualized with UAS-GFP and UAS-mCD8::GFP reporters and anti-GFP antibody (green). In both

panels, neuropils are visualized with nc82 antibody (grayscale). The labial nerve projects to the SOG (blue dotted lines indicate the bound-

ary), which is located in the ventral side of the brain. Antennal lobes (AL; yellow dotted line) and the antennal mechanosensory centers

(AMMC; violet dotted line) are also indicated. The white dotted rectangles indicate the area shown in Figures 6 and 15. J: Schematic dia-

gram of the neuropils around the SOG. The SOG is situated below the oesophagus (OES). The AL and AMMC occupy the area above and

dorsolateral to the SOG, respectively. Sensory neuron axons via the labial nerve (LbN) project to the primary gustatory center (PGC) as

well as to the AL via the antenno-suboesophageal tract (AST). Sensory neuron axons via the pharyngeal and accessory pharyngeal nerves

(PhN) enter the anteriormost and lateral part of the SOG. K: Schematic diagram of the human and fly gustatory neural circuits. The fly

GRNs send their axons directly to the primary gustatory center, whereas the human TRCs send taste information indirectly via the taste

sensory neurons. Detailed neural structures in the primary gustatory center are yet to be elucidated in both human and flies. Scale bar ¼50 mm in A; 100 lm in B,C; 50 lm D (applies to D,E) F, and H (applies to H,I).

Primary gustatory center of Drosophila

The Journal of Comparative Neurology | Research in Systems Neuroscience 4149

saltiness (Huang et al., 2006; Ishimaru et al., 2006; Chan-

drashekar et al., 2010). TRCs express defined combina-

tions of these gustatory receptors and are supposed to

receive particular modalities of taste stimuli (Chandrashe-

kar et al., 2010; reviewed in Chandrashekar et al., 2006;

Ishimaru, 2009; Yarmolinsky et al., 2009). The taste neu-

rons connecting the TRCs and NST are activated prefer-

entially by one of the taste modalities (Ninomiya et al.,

1984; Yoshida et al., 2006).

In the olfactory systems of both mammals and insects,

axons of the olfactory sensory neurons that express the

same olfactory receptor gene terminate in a distinct small

zone of the primary olfactory center, called the glomeru-

lus, to form an odor map (Vassar et al., 1994; Mombaerts

et al., 1996; Gao et al., 2000; Vosshall et al., 2000).

Because mammalian TRCs do not by themselves inner-

vate the primary gustatory center (Fig. 1K), it is difficult to

examine the taste sensory map by tracing the information

pathways from specific types of TRCs (Sugita and Shiba,

2005; Damak et al., 2008; Ohmoto et al., 2008). The

insect gustatory system has an advantage in this respect,

because their GRNs send axons directly to the brain (Fig.

1K).

Projection patterns of GRNs from the mouthparts or

antennae have been analyzed in various insects including

cockroach (Nishino et al., 2005), honeybee (Rehder,

1989), mosquito (Ignell and Hansson, 2005), and flies

(Nayak and Singh, 1985; Yetman and Pollack, 1986;

Edgecomb and Murdock, 1992; Shanbhag and Singh,

1992). GRNs on the fly’s labellum show various charac-

teristic projection patterns to distinct areas of the PGC

(Nayak and Singh, 1985). The Drosophila gustatory recep-

tor (Gr) genes were identified as a large multigene family

of seven-transmembrane receptors expressed in the

GRNs. Some of them were shown to be responsible for

the detection of sugars and bitter compounds (reviewed

in Amrein and Thorne, 2005; Montell, 2009; Yarmolinsky

et al., 2009). Targeted expression of green and red fluo-

rescent proteins (GFP and DsRed, respectively) under

control of the promoter sequences of these Gr genes

revealed that sugar-detecting and bitter-sensing GRNs in-

nervate different regions of the PGC (Thorne et al., 2004;

Wang et al., 2004; Marella et al., 2006). Other GRNs nec-

essary for the detection of water or carbon dioxide (CO2)

have also been identified by using GAL4 enhancer-trap

strains (Inoshita and Tanimura, 2006; Fischler et al.,

2007).

The gustatory neural circuits described so far in the

Drosophila brain, however, are likely to represent only a

subset of the entire sensory neurons involved in taste

detection. The water- and CO2-responsive GRNs identi-

fied with the enhancer-trap system had not been labeled

with any reporters driven by the promoters of known Gr

genes, suggesting that a reverse-genetic approach to

drive expression of reporters using genome information

would not reveal the whole repertoire of GRNs. Previously

unlabeled GRNs might be identified with a forward-

genetic approach by screening a large collection of mo-

lecular markers for visualizing specific neurons, such as

GAL4 enhancer-trap strains.

Another difficulty for the systematic mapping of GRN

projection targets was the lack of clear neuropil bounda-

ries in the candidate area of the PGC. Glomeruli in the pri-

mary olfactory center of insects, the antennal lobe, are

clearly separated by glial processes; each glomerulus can

be visualized with antibodies such as nc82, which labels

synaptic structures (Laissue et al., 1999). In contrast, nei-

ther clear glial boundaries nor demarcated neuropil struc-

tures are observable in the area of the SOG that suppos-

edly houses the PGC. To compare projections of different

neurons labeled with GAL4 drivers, a new technique is

required for visualizing landmark structures reproducibly

without relying on the GAL4/UAS system.

To surmount this problem, we utilized the LexA tran-

scriptional system, which can drive reporter gene expres-

sion independent of the GAL4/UAS system (Lai and Lee,

2006). We screened a newly established collection of

LexA::VP16 (LexAV) enhancer-trap strains (Endo et al. in

preparation) to obtain a marker that can visualize projec-

tions of a large subset of GRNs. By simultaneously visual-

izing neurons labeled with GAL4 and LexAV strains, we

mapped the projection targets of the GRNs and taste-

associated mechanosensory neurons on the labellum and

identified 11 zones in the PGC, as well as three more

zones that the pharyngeal and tarsal GRNs specifically

contribute to.

MATERIALS AND METHODS

Experimental animalsFlies were raised on standard yeast/cornmeal/agar

medium. Female flies aged 5–10 days after eclosion at

25�C were examined in all experiments. The transgenic

GAL4-driver strains used for visualizing specific neurons

with the GAL4/UAS system are described below (Brand

and Perrimon, 1993). In total 3,939 GAL4 enhancer-trap

strains (MZ series: Ito et al., 1995; NP series: Yoshihara

and Ito, 2000; Hayashi et al., 2002) were screened to

identify the strains that label selective subsets of GRNs.

The six strains selected and used in this study are as fol-

lows: LB1: NP7506, LB2: NP2113, LB3: NP1513, LB4:

NP171, LB5: NP1371, and LB6: NP136.

Gr2a-GAL4, Gr32a-GAL4, Gr47a-GAL4, and Gr66a-

GAL4 (Scott et al., 2001; Wang et al., 2004), GAL4-

NP1017 (Inoshita and Tanimura, 2006), and GAL4-E409

(Fischler et al., 2007) strains were used for driving

Miyazaki and Ito


reporter expression in already identified GRNs. Or83b-

GAL4 (Wang et al., 2003) was used for labeling the olfac-

tory receptor neurons (ORNs) in the maxillary palp.

The following transgenic fly strains were used for dou-

ble-labeling other GRNs in combination with the GAL4/

UAS system. The LexA::VP16 (LexAV) enhancer-trap

strains (Endo et al., in preparation) were screened to visu-

alize a large subset of the GRNs in the SOG with the LexA

transcriptional system (Lai and Lee, 2006). Transgenic

flies that have multiple copies of GFP sandwiching inter-

nal ribosomal entry sites (IRES) under control of the Gr5a

and 66a promoters (i.e. Gr#-GFP-IRES-GFP-IRES-GFP;

referred to as Gr#-GFP) (Wang et al., 2004; Fischler et al.,

2007) were used for visualizing the respective type of the

GRNs.

UAS-reporter strains used were as follows: UAS-GFP

S65T (T2 strain, gift from Barry Dickson; Ito et al., 1997)

and UAS-mCD8::GFP (LL6 strain; Lee and Luo, 1999) for

visualizing the general morphology of the GAL4-express-

ing cells, and a fly strain carrying both UAS-GFP S65T and

UAS-mCD8::GFP for visualizing both thick neural fibers

and varicosities (labeled strongly with cytoplasmic GFP

S65T) and thin neural fibers (labeled strongly with mem-

brane-bound mCD8::GFP; Ito et al., 2003). A strain bear-

ing both UAS-neuronal synaptobrevin::GFP (n-syb::GFP; Ito

et al., 1998; Estes et al., 2000) and UAS-DsRed S197Y

(Verkhusha et al., 2001) was used for labeling the presyn-

aptic sites and overall neural fibers simultaneously. Both

lexAoperator-rCD2::GFP (lexAop-rCD2::GFP; Lai and Lee,

2006) and lexAoperator-GFP (lexAop-GFP, gift from Take-

shi Awasaki and Tzumin Lee) were used in combination

with UAS-DsRed S197Y for double- labeling GAL4- and

LexAV-expressing neurons.

ImmunohistochemistoryAntibodies used in this study were as follows. Anti-

ELAV (embryonic lethal, abnormal vision; rat monoclonal,

Developmental Studies Hybridoma Bank, Iowa City, IA,

#RAT-Elav-7E8A10, used at 1:250 dilution) was used for

labeling neuronal cell bodies. ELAV is a nuclear protein

expressed in most postmitotic neurons in central and pe-

ripheral nervous systems of Drosophila (Robinow and

White, 1988, 1991). The antibody was raised against the

first 260 amino acids of phage T7 gene 10 fused to the

entire 483-amino-acid ELAV protein produced in Esche-

richia coli BL21 plysS. It labeled nuclei of the neurons

located in all regions of the cell body cortex in the fly

brain (data not shown). On the surface of the labellum,

the appropriate number of neuronal nuclei (i.e., five or

three for a labellar gustatory bristle, two for a taste peg,

and one for a mechanosensory bristle) at the root of each

sensillum was visualized with this antibody (data not

shown). We observed no other labeling in the brain and

on the surface of the labellum with the antibody (data not

shown).

Anti-DsRed (Living Colors DsRed Polyclonal Antibody;

Clontech, Mountain View, CA, ref. 632496, used at 1:500

dilution) was used for enhancing the signals of UAS-

DsRed. This antibody is rabbit antisera raised against

DsRed-Express, a variant of Discosoma sp. red fluorescent

protein, and recognizes DsRed-express, DsRed-Monomer,

and both N- and C-terminal fusion proteins containing

these DsRed variants in mammalian cell lysate.

Anti-GFP (rabbit polyclonal, Invitrogen, La Jolla, CA,

#A11122, used at 1:1,000 dilution; and mouse monoclo-

nal, Roche Diagnostics, Indianapolis, IN, #1814460, used

at 1:300 dilution) was used for enhancing the GFP signals

of UAS-GFP, lexAop-rCD2::GFP, lexAop-GFP, and Gr#-

GFP. Rabbit anti-GFP was raised against GFP isolated

directly from Aequorea victoria and purified by ion

exchange to remove nonspecific immunoglobulins.

Mouse anti-GFP was a mixture of two high-affinity mono-

clonal antibodies that were obtained by immunizing mice

with partially purified recombinant GFP as immunogen

and selected for their superior performance in detecting

GFP and GFP fusion proteins. The specificity of anti-GFP

and anti-DsRed antibodies was checked in situ by con-

firming the lack of immunolabeling in the brain and

mouthpart of the animals that do not express GFP and

DsRed reporters (Ito et al., 2003; our unpublished data).

Synaptic areas were visualized with mouse monoclonal

antibodies anti-Bruchpilot nc82 (Hofbauer, 1991; Wagh

et al., 2006; gift from E. Buchner and A. Hofbauer, used

at 1:250 dilution) or anti-Synapsin (anti-SYNORF1) 3C11

(Klagges et al., 1996; Godenschwege et al., 2004; Devel-

opmental Studies Hybridoma Bank; used at 1:1,000 dilu-

tion). The anti-Bruchpilot nc82 antibody is an IgG pro-

duced by a hybridoma clone from a large library

generated against Drosophila heads (Hofbauer, 1991). It

recognizes the ubiquitously expressed active zone pro-

tein, Bruchpilot, which forms protein bands of 170 and

190 kDa in Western blots of homogenized Drosophila

heads (Wagh et al., 2006). The immunoreactive signal dis-

appears in the tissue as well as in the Western blots if the

bruchpilot gene is knocked down, and an additional band

is detected specifically if GFP-tagged Bruchpilot is

expressed in a pan-neuronal manner (Wagh et al., 2006).

The anti-Synapsin 3C11 antibody is an IgG produced

by a hybridoma clone, which is derived from spleen cells

from immunized Balb/C-mice and myeloma cells. The

mice were immunized with GST-tagged fusion protein

translated from the 50 segment of Drosophila synapsin

cDNA (Klagges et al., 1996). Synapsin is an abundant syn-

aptic phosphoprotein, which is supposed to be associ-

ated with neurotransmitter release by maintaining a syn-

aptic vesicle pool in presynaptic boutons (Akbergenova



and Bykhovskaia, 2007). Mutant flies lacking synapsin, in

which no immunoreactive signal is observed in the brain

neuropils, show normal morphology of brain structure

and excitatory potentials, whereas complex behaviors

such as learning are inhibited in these flies (Godensch-

wege et al., 2004).

The nc82 and 3C11 antibodies give similar labeling

results (Kazunori Shinomiya, unpublished observation),

but for nc82 labeling, an additional 30-minute incubation

with 2% Triton-X 100 in phosphate-buffered saline (PBS;

pH 7.4 at 25�C) prior to antibody labeling and extended

incubation time with the primary antibody were required

to obtain better results.

Secondary antibodies used in this study were as

follows: Alexa Fluor 488-conjugated anti-mouse IgG (Invi-

trogen; used at 1:250 dilution) and anti-rabbit IgG (Invitro-

gen; #A11008, 1:1000), Alexa Fluor 568-conjugated

anti-rabbit IgG (Invitrogen, 1:250), and Alexa Fluor 647-

conjugated anti-mouse IgG (Invitrogen, 1:250) and anti-rat

IgG (Invitrogen; #A21247, 1:250). No labeling was seen

when Alexa Fluor-conjugated anti-mouse/rabbit/rat IgGs

were used for labeling tissues without primary antibodies.

Imaging of the labella and maxillary palpsFor the second step of screening, about 100 candidate

GAL4 enhancer-trap strains, screened from a collection

of 3,939 strains, were crossed with the flies carrying both

UAS-GFP and UAS-mCD8::GFP. Female F1 progeny 5–10

days after eclosion (at 25�C) were anesthetized with car-

bon dioxide gas prior to decapitation and rinsed briefly in

ethanol. Then the mouthparts were cut off from the heads

at their bases in PBS and observed directly, without fixa-

tion, by using LSM 510 confocal microscopes (Carl Zeiss,

Gottingen, Germany) with a water-immersion 40� C-Apo-

chromat (NA 1.2) objective lens.

For more detailed analyses (Figs. 2–5, 7, 12, S1, S3),

the dissected mouthparts were cut straight at the midline

in PBS and fixed with 4% formaldehyde (methanol free,

ultra pure EM grade, Polysciences, Warrington, PA) in

PEM (100 mM PIPES, 2 mM EGTA, 1 mM MgSO4, pH

6.95) for 30 minutes at 4�C. Samples were rinsed once

for 5 minutes with PBS, twice for 15 minutes with PBS

containing 0.5% Triton X-100 (PBT), and subsequently

incubated in the blocking solution (BS), which contains

10% normal goat serum (Vectastain, Vector, Burlingame,

CA) in PBT, for 1 hour, then in a mixture of primary anti-

bodies in BS overnight. Samples were rinsed three times

for 15 minutes with PBT and incubated with a mixture of

secondary antibodies in BS overnight. After three 15-

minute rinses with PBT and a 5-minute rinse with PBS,

samples were cleared with a 2-hour incubation in 50%

glycerol (for fluorescence microscopy; Merck, Darmstadt,

Germany) in PBS and subsequent overnight incubation in

80% glycerol. Maxillary palps were then cut off from the

main part of the mouth including labella. The palps and

labella were mounted on slides with 0.054-mm-thick poly-

propylene tape and 0.2-mm-thick polyvinyl chloride elec-

trical insulating tape as spacers, respectively.

Mouse monoclonal anti-GFP and rabbit polyclonal anti-

DsRed antibodies were used for enhancing the reporter

signals, and rat monoclonal anti-ELAV antibody was used

for labeling neuronal cell bodies. Alexa Fluor 488-conju-

gated anti-mouse IgG, Alexa Fluor 568-conjugated anti-

rabbit IgG, and Alexa Fluor 647-conjugated anti-rat IgG

were used for these primary antibodies.

Dissection and immunolabeling of the brainsThe axon terminals in the brains shown in Figures 6,

15A–F, S2, and S4A–F were examined without immuno-

labeling. The brains were dissected from the decapitated

head capsule in PBS and fixed in 4% formaldehyde in PEM

for 2 hours at 4�C. After three 15-minute rinses with PBS,

samples were incubated with 50% glycerol in PBS for 2

hours and cleared with 80% glycerol overnight.

For immunolabeling of the brain (Figs. 1, 7–15, S3, S4),

dissected and fixed samples were processed in the same

manner as explained in the previous section. For Figure

1H, rabbit polyclonal anti-DsRed antibody was combined

with mouse monoclonal nc82 antibody, with Alexa Fluor

568-conjugated anti-rabbit IgG and Alexa Fluor 647-con-

jugated anti-mouse IgG, to visualize the labeled neurons

and all the neural cell bodies, respectively. For Figure 1I,

rabbit polyclonal anti-GFP antibody was combined with

mouse monoclonal nc82 antibody, with Alexa Fluor 488-

conjugated anti-rabbit IgG and Alexa Fluor 647-conju-

gated anti-mouse IgG, to visualize the labeled neurons

and all the neural cell bodies, respectively. For Figures 7–

14, 15G, S3, and S4G, mouse monoclonal anti-GFP and

rabbit polyclonal anti-DsRed antibodies were used for

enhancing the GFP and DsRed signals, respectively, with

Alexa Fluor 488-conjugated anti-mouse IgG and Alexa

Fluor 568-conjugated anti-rabbit IgG.

Imaging of the labeled cells and neuralfibers

Confocal serial optical sections at 0.27–1.2-lm inter-

vals were taken by using LSM 510 confocal microscopes

(Carl Zeiss) with 20� Plan-Apochromat (NA 0.75), water-

immersion 40� C-Apochromat (NA 1.2), and oil-immer-

sion 63� (NA 1.4), and 100� (NA 1.4) Plan-Apochromat

objective lenses. Sections of the brains were taken from

the anterior side.

Single sections and stacks of multiple serial sections

were generated with the software of the LSM510 confo-

cal microscope. For 3D reconstruction (Figs. 1H, 1I, 2, 4,

Miyazaki and Ito


6, 7A–C, 7J, 13, 15, S1–4), confocal datasets were proc-

essed with the 3D reconstruction software Imaris 2.7 (Bit-

plane, Zurich, Switzerland) running on Silicon Graphics

(Fremont, CA) Octane2 or Fuel workstations by using the

‘‘ray tracing’’ algorithm with the transparency parameter

set at 50–97%, depending on the condition of the dataset.

3D schematic models (Figs. 11C–F, 14G–I) were con-

structed based on the labeling data of the brain samples

by using Amira software (Visage Imaging, Berlin, Ger-

many). The size, resolution, and contrast of the final

images were adjusted with Photoshop CS3 software

(Adobe Systems, San Jose, CA).

Erasure of the signals of other labeled cellsthan the GRNs

Enhancer-trap strains tend to label various cell types in

the brain. To visualize the morphology of only the selected

GRNs more clearly, in some cases we erased the signals of

other labeled cells from the confocal datasets (Figs. 6, 7G–

I, 9, 10, 12H–L, 13, 14, S2). To do this efficiently, the LSM

dataset was converted to a series of TIFF-format images

and imported to Photoshop software (Adobe Systems) run-

ning on a Windows computer. Each confocal section was

assigned to a distinct layer. By comparing the labeling pat-

terns between neighboring layers (i.e., sections), the cell

bodies and fibers of the labeled GRNs and other labeled

cells were distinguished. In each section, signals attributed

to the latter category were selected and painted in black

(i.e., background color). The resulting images were re-con-

verted to PNG-format files and imported to LSM510 soft-

ware to make stacked projections (Figs. 7, 9, 10, 12–14),

and further exported to Imaris 2.7 software to generate 3D

reconstructed micrographs (Figs. 6, S2). The reconstruction

images without erasure of other signals are presented in

Figures 15 and S4.

RESULTS

Identification of GAL4 enhancer-trap strainsthat label GRNs

To obtain molecular markers that label distinct subsets

of GRNs, we screened a collection of 3,939 GAL4

enhancer-trap strains (NP- and MZ-series; Ito et al., 1995;

Yoshihara and Ito, 2000; Hayashi et al., 2002). As the first

screening, we searched for the GAL4 lines that label neu-

ral fibers projecting via the labial nerve to the SOG. About

100 candidate strains were identified. Many of them

seemed to label not only GRNs in the labella but also

ORNs in the maxillary palps, which also project to the

SOG via the same labial nerve (Stocker and Schorderet,

1981). To exclude such lines, we crossed the candidate

lines with the flies carrying UAS-GFP and examined the

expression patterns in the labella and maxillary palps.

Among the 100 strains, 81 were found to label cells in the

labella, but 57 of them also showed strong expression in

the maxillary palps. The remaining 24 strains labeled cells

in the labella preferentially, but many of them also labeled

various non-neural cells such as epidermal cells. Because

labeling in these cells may disturb precise analyses of the

position, number, and morphology of the GRNs, we

excluded 18 strains that fall in this criterion. Finally, we

identified six strains that label candidate GRNs rather

specifically and subjected them to detailed examination.

We renamed these lines GAL4-LB1 to GAL4-LB6 for sim-

plicity (LB for ‘‘labellum’’; among them, LB2 and LB5 la-

beled a few non-neuronal cells inside the labellum; see

the next section).

Distribution and number of the labellarneurons labeled with each strain

The labellum consists of two regions. Its lateral surface

has a hemispherical shape (Fig. 1D), on which there are

approximately 30 gustatory sensilla (arrowheads in Fig.

1D) and two mechanosensory bristles (arrows in Fig. 1D)

per side (Nayak and Singh, 1983; Shanbhag et al., 2001).

The mechanosensory bristles are located in the most dor-

solateral area of the labellar surface and are shorter than

the gustatory sensilla. Each gustatory sensillum houses

two or four GRNs and one mechanosensory neuron (Fig.

1G), whereas each mechanosensillum has just one

mechanosensory neuron at its root. The labellum also has

a deep recess along the midline (in Fig. 1E, the labellum

of this sample was cut at the midline). The inner (medial)

surface of this recessed area has groove structures called

pseudotracheae (arrows in Fig. 1E). Taste pegs lie

between these grooves (arrowheads in Fig. 1E). We found

30–40 taste pegs on each side. Each taste peg contains

one GRN and one mechanosensory neuron (Nayak and

Singh, 1983; Shanbhag et al., 2001). In the medial recess

there are no sensilla that are specialized for mechanosen-

sation. Cell bodies of the GRNs are not always situated

right at the position of each bristle or taste peg; the neu-

rons send thin dendrites toward the inner lumen of these

sensory organs. In addition, there are non-neural support-

ing cells around the neurons.

To examine the types of cells labeled with LB1–6

strains, we visualized the GAL4-expressing cells by using

the UAS-DsRed reporter and all the neurons with the anti-

ELAV antibody. The six LB strains showed characteristic

distribution of the labeled cells in the labellum (Table 1,

Fig. 2; 3D stereograms in Fig. S1). LB1 labeled neurons in

about two-thirds of the medial taste pegs (Figs. 2A, 3H)

as well as neurons at the root of the two mechanosensory

bristles on the lateral surface (arrows in Figs. 2A, 3A).

LB2 labeled neurons in about two-thirds of the medial

taste pegs (Figs. 2B, 3I) as well as non-neuronal cells in



the lateral part (Figs. 2B, 3B, magenta cells). LB3 and LB4

labeled neurons in both lateral sensilla and medial taste

pegs (Figs. 2C,D, 3C,D,J,K). LB5 and LB6 labeled only a

few neurons in the lateral sensilla (Figs. 2E,F, 3E,G). LB5

also labeled some non-neuronal cells in the lateral part

(Figs. 2E, 3F, magenta cells).

Each lateral sensillum houses either three (two chemo-

sensory and one mechanosensory) or five (four chemo-

sensory and one mechanosensory) neurons. All four

strains that labeled neurons in the lateral sensilla (LB3-6)

visualized only one neuron per sensillum. The nuclei of

the neurons of both lateral sensilla and medial taste

pegs, visualized with the anti-ELAV antibody, showed a

rather constant size of approximately 3 lm in diameter.

The diameters of the neural cell bodies, in contrast, were

more diverse. Whereas all the neurons in the medial taste

pegs and the lateral sensilla neurons labeled with LB3–5

strains had cell bodies that were 5–10 lm in diameter

(Fig. 3A,C–E,H–K), the somata of the lateral sensilla neu-

rons labeled with LB6 were as large as 15 lm (Fig. 3G).

Initial screening of the GAL4 enhancer-trap lines, which

was performed by observing unfixed samples, identified

no signals in the maxillary palps of the LB1–6 lines. More

sensitive microscopy with antibody labeling, however,

identified faint labeling of some neurons in the maxillary

palps of LB1–5 (Fig. 4A–E). LB6 did not visualize any neu-

rons in this appendage (Fig. 4F).

Labeling of the mechanosensory-bristle neurons on the

lateral surface of the labellum with the LB1 strain led us

to the notion that the neurons of the medial taste pegs la-

beled in this line may also be mechanosensory rather

than gustatory. To test this hypothesis, we compared the

labeling patterns of LB1 and the E409 strain, a GAL4

enhancer-trap line that labels taste-peg GRNs involved

with reception of carbonated water (Fischler et al., 2007).

Both LB1 and E409 strains labeled four to six neurons per

one row of taste pegs (Fig. 5A,B). When the two driver

lines were combined, the number of labeled neurons

almost doubled (Fig. 5C). This strongly suggests that LB1

and E409 label essentially exclusive sets of neurons.

To confirm their different identities further, we per-

formed double-labeling experiments on the two GAL4

strains with a LexAV enhancer-trap strain, NV4, which

labels a large population of GRNs (see below). There were

few neurons labeled with both LB1 and NV4 (Fig. 5D,F),

showing that the two lines label mutually exclusive sets

of cells. In contrast, most of the cells labeled with E409

and NV4 were overlapping (Fig. 5E,G). Because each taste

peg has one chemosensory neuron and one mechanosen-

sory neuron (Falk et al., 1976; Nayak and Singh, 1983),

we concluded that E409 and LB1 label chemosensory

and mechanosensory neurons, respectively. Although

both lateral gustatory sensilla and medial taste pegs are

associated with mechanosensory cells, only the latter

group was labeled with LB1. LB1 also labeled mechano-

sensory bristles on the surface of the abdomen, but not

the legs (data not shown). Thus, LB1 labels a distinct sub-

set of mechanosensory neurons.

Overall projection patterns of the labeledneurons

The neurons in the labellum project their axons to the

brain via the labial nerve. To analyze their projection

TABLE 1.

Summary of Labeled Cells1

Lateral sensilla

Medial taste pegs Projection targets

No.

Chemosensory Mechanosensory

Min Med Max Min Med Max Min Med Max AMS PMS LS

LB1 0 1 2 1 2 2 15 19.5 31 AMS2,3 LS3 6LB2 0 0 2 0 0 0 19 23 32 AMS1 PMS1,2,3 LS1,2 5LB3 3 4 6 0 0 0 7 8 9 AMS1 PMS1,2,3 3LB4 12 18 20 0 0 0 0 2 7 PMS1 4LB5 2 2 4 0 0 0 0 0 3 PMS1 5LB6 3 3 4 0 0 0 0 0 0 PMS1,2 5NV4 þ 0 þ AMS1 PMS1,2,3 LS1,2NP1017 þ 0 þ AMS1 PMS 4,5E409 0 0 þ AMS1Gr66a þ 0 0 PMS1,2,3Gr32a þ 0 0 PMS1,2,3Gr47a þ 0 0 PMS 2,3Gr5a þ 0 0 PMS 4,5

1The numbers of cells visualized with GAL4 enhancer-trap strains and UAS-DsRed reporter were counted in the lateral sensilla and medial taste

pegs of the labellum. The minimum, median, and maximum of the numbers of the labeled cells are shown. Only labeling patterns are indicated for

the already known types of GRNs. The fifth through seventh columns summarize the projection targets of each GRN type. In the rightmost column,

No. indicates the number of samples counted.

Miyazaki and Ito


patterns in the primary gustatory center (PGC), we then

observed their axons in the brain. Cytoplasmic GFP re-

porter visualizes thick neural fibers and varicosities

strongly, but it labels thin fibers relatively weakly because

of the small amount of cytoplasm within them. In con-

trast, membrane-bound GFP visualizes thin fibers

Figure 2. (legend on page 4156)



strongly, but thick fibers and varicosities are less pro-

nounced because the ratio between the surface and cyto-

plasmic volume is smaller in these thick structures (Ito

et al., 2003). To visualize thick and thin processes of the

GAL4-expressing neurons equally clearly, we used a re-

porter strain that carries both UAS-GFP and UAS-

mCD8::GFP.

LB1–3 labeled axons that upon entering the SOG bent

anteriorly to terminate in the anteriormost area of the

SOG (Fig. 6A–C; 3D stereograms in Fig. S2). LB2–6 la-

beled axons that projected straightly upward in the ante-

rior area of the SOG (Fig. 6C–F). LB1 and 2 also labeled

axons that bent posteriorly to terminate in the middle

area of the SOG (Fig. 6A,B). The three regions of the la-

beled terminals were correlated with the anterior maxil-

lary sensory, posterior maxillary sensory, and labellar sen-

sory centers in the recently proposed systematic

nomenclature of the Drosophila brain (Ito et al., in prepa-

ration). We therefore named these branches anterior

maxillary sensory (AMS), posterior maxillary sensory

(PMS), and labellar sensory (LS) branches. LB1 labeled

neurons projecting to the AMS and LS branches, LB2 to

the AMS, PMS, and LS branches, LB3 to the AMS and

PMS branches, and LB4–6 exclusively to the PMS branch

(Fig. 6A2–F2). (Many LB strains also labeled neurons in

the brain that are not associated with the GRNs. Signals

of such neurons were erased from the figures for clarity.

Original images without erasure of such signals are

shown in Figs. 15 and S4.)

Axon terminals of the labeled neurons exhibited vari-

ous projection patterns within each branch. In the AMS

branch, axons labeled with LB2 and LB3 projected to the

lateral region of the SOG (Fig. 6B1,C1), whereas LB1 la-

beled neurons projecting not only to the lateral area but

also toward the medial area, which probably extended to

the contralateral SOG (Fig. 6A1). The PMS branch la-

beled with LB4 and LB5 was confined within the ipsilat-

eral area of the SOG (Fig. 6D1,E1), but the area of the

PMS branch labeled with LB2, 3 and 6 spread to the

medial region and crossed the midline (Fig. 6B1,C1,F1).

These data suggest that neurons labeled with each LB

line arborize in specific areas of the PGC. However,

slight variability among individuals, distortions in sample

preparation, and fluctuation of the sample orientation in

confocal microscopy prevented us from determining the

precise projection areas of each neuron type.

A LexA::VP16 enhancer-trap strain thatvisualizes a landmark structure in the SOG

Clearly and reproducibly identifiable landmarks are in-

dispensable for examining precise projection patterns of

neurons within a particular neuropil. In the primary olfac-

tory center of insects, the antennal lobe, synaptic

markers such as anti-Bruchpilot nc82 and anti-Synapsin

antibodies provide distinct labeling of individual glomeruli

(Laissue et al., 1999; our unpublished data). The land-

mark structures identified have been used successfully

for determining the projection sites of the neurons in this

neuropil. These antibodies unfortunately give only

obscure labeling pattern in the SOG, where the PGC

exists. The three branches labeled with the LB1–6 lines

are only vaguely and incomprehensively visible with any

of the known antibodies. A novel technique is required for

visualizing landmark structures in the SOG independent

of the GAL4/UAS system.

A dual binary transcriptional system that works in Dro-

sophila melanogaster was reported recently (Lai and Lee,

2006). In this system, an Escherichia coli-derived tran-

scription factor (LexA) activates the expression of the re-

porter genes under control of its target sequence, lexAop-

erator (lexAop), without interfering with the GAL4/UAS

system. To label various types of neurons with this sys-

tem, we have created a collection of about 320 Lex-

A::VP16 (LexAV) enhancer-trap strains (Endo et al., in

preparation). In screening these lines, we identified one

strain, NV4, which labels a relatively large subset of the

GRNs that collectively project to all three branches of the

PGC (Fig. 7A–C,G–J; 3D stereograms in Fig. S3). By dou-

ble-labeling a large subset and specific subsets of the

GRNs with LexAV-NV4 and GAL4-LB1 through 6, we were

able to map the projection sites of each subtype of GRNs

in the overall framework of the PGC.

Figure 2. GAL4 enhancer-trap strains that label neurons in the labellum. First (A1–F1) and second (A2–F2) columns: 3D reconstruction of

the labellum showing the cells that express UAS-DsRed under the control of each GAL4 driver strain. Red-cyan 3D stereograms are avail-

able in Supplementary Figure S1. Third (A3–F3) and fourth (A4–F4) columns: Superposition of the labeled cells (yellow, visualized with

UAS-DsRed expression and anti-DsRed antibody), all the neurons (blue, anti-ELAV antibody), and cuticle (gray, autofluorescence). First and

third columns show frontal views (dorsal up, lateral to the right). Dotted lines indicate the borders between the lateral surface and medial

recess regions of the labellum. Second and fourth columns represent lateral views (dorsal up, anterior to the left). The areas shown are

indicated as a rectangle in Figure 1D. A,B: LB1 and LB2 label neurons in the medial taste pegs. In addition, LB1 labels neurons of the two

mechanosensilla on the lateral surface of the labellum (arrow). Although LB2 also labels some cells on the lateral surface (B), they are

ELAV-negative and therefore not neurons (see Fig. 3). C,D: LB3 and LB4 label neurons in both the lateral sensilla and medial taste pegs.

E,F: LB5 and LB6 label neurons only in the lateral sensilla. Scale bar ¼ 50 lm in A1 (applies to A1–F4).

Miyazaki and Ito


Figure 3. Neural identity of the GAL4-expressing cells. High-magnification views of the single confocal sections of the data shown in Fig-

ure 2. GAL4-expressing cells revealed with UAS-DsRed and anti-DsRed antibody (first columns, A1–K1), all the neurons labeled with anti-

ELAV antibody (second panels, A2–K2), and merged image of the two channels (third panels, A3–K3; magenta, DsRed; green, anti-ELAV).

Arrows indicate neurons labeled with GAL4 enhancer-trap strains. A: LB1 labels neurons at the root of the mechanosensilla on the lateral

surface of the labellum. B: LB2 labels non-neural cells on the lateral surface. C–G: LB3–6 label neurons on the lateral sensilla. LB3–5 label

small neurons with 5–10-lm diameter (C–E), whereas LB6 labels large neurons with ca. 15-lm diameter (G). LB5 also labels some non-

neural cells on the lateral surface of the labellum (F). H–K: LB1-4 label neurons on the medial taste pegs. Their diameters are 5–10 lm.

Scale bar ¼ 10 lm in A3 (applies to A1–K3).



Unlike the six GAL4 strains used in this study, LexAV-

NV4 was much less specific to GRNs. In addition to

GRNs, NV4 labeled many ORNs in the antenna, ORNs and

mechanosensory neurons in the maxillary palp (Fig. 7D–

F), and Johnston’s organ neurons that project from the

base of the antenna to the antennal mechanosensory and

motor center (AMMC; data not shown). NV4 therefore la-

beled a large subset of the peripheral sensory neurons.

Within the brain, the strain labeled terminals of the ORNs

in the antennal lobe, neural processes extending from the

AMMC to the space between the PMS and LS branches

of the PGC, neurons of the optic lobe, and mushroom

body neurons innervating the a0/b0 lobes (data not

shown).

Because ORNs in the maxillary palp and GRNs in the

labellum project through the same labial nerve, a ques-

tion arises of whether they project to the same area of

the brain. The maxillary palp ORNs are reported to project

to the AL via the antenno-suboesophageal tract (AST),

which runs just lateral to the PMS branch of the PGC (Fig.

1J). To confirm this hypothesis, we double-labeled the

brain with LexAV-NV4 and Or83b-GAL4 (Fig. 7).

Because the Or83b gene is expressed in most of the

ORNs, Or83b-GAL4 drives expression in most ORNs in

the antennae and maxillary palps. No signal was observed

in the labellum using this driver (Fig. 7B,C), confirming

that it does not label any GRNs.

Both NV4 and Or83b-GAL4 labeled neurons in the max-

illary palp. Of the total 61 (median, minimum 59, maxi-

mum 82; n ¼ 4) neurons labeled with NV4 in the maxillary

palp, 45 (median, minimum 41, maximum 62; n ¼ 4) neu-

rons were also labeled with Or83b (Fig. 7D–F). Thus,

about three-fourths of the NV4-positive maxillary palp

neurons were ORNs, and the remaining 10–20 neurons

were putative mechanosensory neurons.

In the SOG, Or83b-GAL4 labeled the AST exclusively

(Fig. 7H,I). Thus, all the ORNs in the maxillary palp should

project via the AST to terminate in the AL. NV4 labeled

AST as well as the PGC branches (Fig. 7G,I). It is likely

that the maxillary pulp neurons that are labeled with both

NV4 and Or83b-GAL4 should project via the AST. Short of

a reliable technique to visualize specifically the axons of

the NV4-positive/Or83b-negative neurons, the projection

target of the putative mechanosensory neurons in the

maxillary palps remained unknown. Nevertheless, the

Figure 4

Figure 4. Labeling of the GAL4 enhancer-trap strains in the maxil-

lary palps. Left columns (A1–F1): 3D reconstruction of the maxil-

lary palps showing the cells labeled in each GAL4 driver strain.

Lateral view (dorsal up, anterior to the top-left). Right columns

(A2–F2): Superposition of the labeled cells (yellow, UAS-GFP

expression and anti-GFP antibody), all the neurons (blue, anti-ELAV

antibody), and cuticle (gray, autofluorescence). A–E: LB1-5 exhibit

weak labeling of the neurons in the maxillary palp. F: LB6 shows

no expression. Scale bar ¼ 50 lm in A1 (applies to A1–F2).

Miyazaki and Ito


NV4-positive GRNs projecting from the labellum to the

three PGC branches provide useful landmarks required

for the precise mapping of the projection targets of the

GRNs labeled with GAL4 strains (Fig. 7I,J).

As mentioned earlier, LB1–5 showed weak labeling in

the maxillary palp ORNs. Among them, LB3–5 labeled

some fibers in the AST (data not shown). AST were not

visualized in LB1 and 2. Perhaps the expression levels in

these cells might be so weak that not enough GFP mole-

cules were transported to their axons in the AST.

The anterior maxillary sensory branch iscomposed of three zones

Three GAL4 lines, LB1–3, labeled the AMS branch of

the PGC. To analyze the projection sites of the labeled

neurons precisely, we double-labeled these neurons with

LexAV-NV4 (Fig. 8). As described earlier, neurons labeled

with LB2 and 3 terminated in the lateral area of the ipsi-

lateral anteriormost SOG. This area of the AMS branch

overlapped completely with the area labeled with NV4

(Fig. 8B,C). The putative mechanosensory neurons la-

beled with LB1, in contrast, projected to both lateral and

medial areas of the anteriormost SOG. Neither area over-

lapped with the projection targets of the NV4-positive

neurons (Fig. 8A). Thus, the projection targets of the

GRNs in the AMS branch should consist of at least three

zones. Zone AMS1 was contributed by the neurons la-

beled with LB2, LB3, and NV4. The lateral and medial

components of the branch, which were contributed exclu-

sively by LB1-positive neurons, were named zones AMS2

and AMS3, respectively (Fig. 11C,D). Zone AMS2 occu-

pied the lateralmost area of the AMS branch, whereas

zone AMS3 spread in the medial SOG and reached the

midline (inset of Fig. 8A2).

Among the previously identified GRNs, those labeled

with GAL4-NP1017 and GAL4-E409 are known to project

Figure 5. Comparison of the labeling patterns of GAL4-LB1 and GAL4-E409. A–C: Neurons labeled with LB1 (A), E409 (B), and combination

of LB1 and E409 strains (C) on the medial surface of the labellum. GAL4-expressing neuron are visualized with the UAS-GFP reporter and

the anti-GFP antibody. Magnified single confocal sections are shown. Combination of the two drivers (C) labeled a larger number of neu-

rons than those labeled with single drivers (A,B). D,E: Double labeling of GAL4 and LexA::VP16 (LexAV) enhancer-trap strains. The neurons

labeled with GAL4-LB1 (D, magenta) and GAL4-E409 (E, magenta) are shown with the neurons labeled with LexAV-NV4 (green). GAL4- and

LexAV-expressing neurons are visualized with the UAS-DsRed reporter and anti-DsRed antibody and the lexAop-GFP reporter and anti-GFP

antibody, respectively. GAL4-LB1 and LexAV-NV4 label mutually exclusive sets of cells (D), whereas E409 and NV4 label overlapping popu-

lations of neurons (E). F: The numbers of the neurons labeled with either or both LB1 or/and NV4. Left and right edges of the boxes in

the graphs indicate the minimum and maximum numbers of the cells, respectively. Vertical bars indicate the median of the labeled cell

numbers. Few neurons are labeled with both drivers (n ¼ 3). G: The numbers of neurons labeled with either or both E409 or/and NV4.

Most of the neurons labeled with both drivers (n ¼ 6). Scale bar ¼ 5 lm in A (applies to A–E).



Figure 6. Axon terminals of the labeled neurons in the SOG. 3D reconstruction of the anterior part of the SOG. Red-cyan 3D stereograms

are available in Supplementary Figure S2. First columns (A1–F1): Frontal views (dorsal up, lateral to the right). Second columns (A2–F2):

Lateral views (dorsal up, anterior to the left). The areas shown are indicated as rectangles in Figure 1H and I. Both cytoplasmic UAS-GFP

and membrane-bound UAS-mCD8::GFP reporters were expressed to visualize thin and thick fibers clearly (antibodies were not used for

enhancing signals). White dotted lines indicate the outlines of the brain. Signals of the neurons other than the GRNs were erased for

clarity; original data are presented in Figures 15 and S4. A: LB1 labels axons projecting to the anterior maxillary sensory (AMS) and label-

lar sensory (LS) branches. B: LB2 labels axons of the AMS, posterior maxillary sensory (PMS), and LS branches. C: LB3 labels neurons

arborizing in the AMS and PMS branches. D–F: Neurons labeled in LB4–6 project only to the PMS branch. LB4 labels many other neurons

within the SOG that send processes around the terminals of the labeled neurons (green dotted lines in D). G: Schematic diagram of the

projection patterns of the labeled neurons in the SOG (lateral view as in A2–F2;, dorsal up, anterior to the left). Scale bar ¼ 50 lm in A1

(applies to A1–F2).

Miyazaki and Ito


Figure 7. (legend on page 4162)



their axons to the AMS branch (Inoshita and Tanimura,

2006; Fischler et al., 2007). E409 labels CO2-responding

neurons in the medial taste pegs, which project to the

AMS branch. NP1017 labels neurons in both medial taste

pegs and lateral sensilla. The former cells respond to CO2

and project to the AMS branch, whereas the latter

respond to water and terminate in the PMS branch. To

identify the precise targets of the E409- and NP1017-la-

beled neurons in the AMS branch, we double-labeled

these GAL4 lines with LexAV-NV4 and found that their

projection targets both coincided with zone AMS1 (Fig.

8D,E).

The posterior maxillary sensory branch isdivided into five zones

LB2–6 labeled neurons that project to the PMS branch

(Fig. 9). The branch labeled with NV4 consisted of a verti-

cal fiber bundle in the ventrolateral SOG arising from the

entry point of the labial nerve, a horn-like protruding area

extending toward the dorsolateral SOG, and a dorsome-

dial region connecting the PMS branches of both sides.

Projection areas of all the GAL4-LB lines in the PMS

branch were confined within the area visualized with

LexAV-NV4, but neurons of each line projected to specific

subareas. When their projection patterns were compared,

the PMS branch visualized with NV4 could be divided into

three zones. Neural fibers labeled with LB4 and 5 were

distributed only in the horn-like protrusion in the dorsolat-

eral SOG, which we named zone PMS1 (Figs. 9A,B,

11C,E). Neurons labeled with LB2, 3 and 6 also termi-

nated in the more medial region. LB6 did not label the

ventral part of this area (Fig. 9C), whereas LB2 and 3 la-

beled both dorsal and ventral parts (Fig. 9D,E). We there-

fore named the dorsal part zone PMS2 and the ventral

part PMS3 (Fig. 11E). The ventrolateral fiber bundle was

labeled with all five LB lines, but it would be better to

regard this bundle as the tract toward the projection tar-

gets, because none of the lines seem to form synaptic

arborizations in this area (discussed later).

The GRNs expressing seven-transmembrane gustatory

receptor genes are known to send their axons to the PMS

branch. To examine precise projection sites of these

GRNs in the framework of our map, we visualized the bit-

ter-responsive neurons expressing Gr32a, 47a, or 66a

simultaneously with the neurons labeled with LexAV-NV4.

The axons of the GRNs labeled with Gr32a-GAL4 and

Gr66a-GAL4 arborized throughout the region of the PMS

branch visualized with NV4 (i.e., zones PMS1, 2, and 3;

Fig. 9F,G). Those labeled with Gr47a-GAL4 projected to

zones PMS2 and 3 but not to zone PMS1 (Fig. 9H). Thus,

the projection targets of the bitter-responsive GRNs

match exactly with these three zones.

The water-sensitive GRNs labeled with GAL4-NP1017

have their cell bodies in the lateral sensilla and project to

the area of the PMS branch that does not overlap with the

projection terminals of the bitter-sensitive GRNs (Inoshita

and Tanimura, 2006). Indeed, double-labeling with LexAV-

NV4 revealed that the projection area of the NP1017-la-

beled neurons did not overlap with zones PMS1–3 (Fig.

10A). Their terminals were distributed in the region near

the entry point of the labial nerve, around the ventrolateral

vertical fiber bundle through which axons toward zones

PMS1–3 project. We named this area zone PMS4 (Figs.

10B, 11E). In addition to the previously described projec-

tions in zone PMS4, we also found labeled fibers that fur-

ther extended medially to arborize sparsely in the medial

area that is ventral to zone PMS3. We named this projec-

tion area zone PMS5 (Figs. 10B, 11E).

Figure 7. A LexAV enhancer-trap strain that labels landmark structures in the SOG. Frontal view (first columns, A1–C1, dorsal up, lateral

to the right) and lateral view (second columns, A2–C2, dorsal up, anterior to the left) of the labellum, and high-magnification single-section

images of the labeled neurons in the lateral sensilla (third columns, A3–C3) and medial taste pegs (fourth columns, A4–C4). LexAV-

expressing neurons and olfactory neurons were labeled with LexAV-NV4 > lexAop-GFP and Or83b-GAL4 > UAS-DsRed, respectively, and

visualized with anti-GFP and anti-DsRed antibodies. A–C: Expression in the labellum. 3D reconstruction of the cells labeled with LexAV-NV4

(A) and Or83b-GAL4 (B), and superposition (C) of the two signals (green, LexAV-NV4; magenta, Or83b-GAL4) and cuticular autofluorescence

(gray). Neurons in the labellum were labeled with NV4 but not with Or83b. D–F: Labeled cells in the maxillary palp. Stacked images of the

cells labeled with NV4 (D) and Or83b-GAL4 (E), and superposition (F) of the NV4 (green) and Or83b-GAL4 (magenta) signals and cuticular

autofluorescence (gray). Lateral view (dorsal up, anterior to the top-left). Insets show high-magnification view of the labeled cells. NV4

labels Or83b-expressing olfactory neurons as well as Or83b-negative putative mechanosensory neurons in the maxillary palp. G–I: Labeled

axons in the SOG. Stacked images of the neural fibers labeled with NV4 > lexAop-rCD2::GFP (G) and Or83b-GAL4 > UAS-DsRed (H), and

superposition (I) of the NV4 (green) and Or83b-GAL4 (magenta) signals. Frontal view (dorsal up, lateral to the right). Neurons labeled with

NV4 project to the PGC (blue dotted lines in G and I) as well as to the antenno-suboesophageal tract (AST) (arrowheads), whereas Or83b-

expressing neurons project only to the AST. J: 3D reconstruction showing the three branches of the NV4-labeled neurons (arrows; anterior

maxillary sensory [AMS], posterior maxillary sensory [PMS], and labellar sensory [LS]) and the AST (arrowheads). Lateral view (dorsal up,

anterior to the left). Asterisk (*) indicates the signal of unidentified neurons. LbN, labial nerve; aCCF, anterior cerebrocervical fascicle.

Red-cyan 3D stereograms of A, G, and J are available in Supplementary Figure S3. Scale bar ¼ 50 lm in A1 (applies to A1–C2), D (applies

to D–F), and G (applies to G–J); 10 lm in A3 (applies to A3–C4) and D inset (applies to D–F insets).

Miyazaki and Ito


Figure 8. Projection patterns of the labeled neurons in the anterior maxillary sensory (AMS) branch. Neurons labeled with each GAL4

strain were visualized with UAS-DsRed reporter, whereas the overall structure of the PGC was visualized with LexAV-NV4 > lexAop-

rCD2::GFP. Anti-GFP and anti-DsRed antibodies were used to enhance signals. Stacked images of the anterior area of the SOG are shown.

Single-channel images of the neurons labeled with each GAL4 line (first and third columns; A1–E1, A3–E3), superposition of the neurons

labeled with the GAL4 lines (magenta) and NV4 (green) (second and fourth columns; A2–E2, A4–E4). Insets show the schematic diagrams

of the projection patterns. (Dotted lines indicate the projection areas of the NV4-labeled neurons.) First and second columns: frontal view

(dorsal up, lateral to the right), third and fourth columns: lateral view (dorsal up, anterior to the left). A: The projection areas labeled with

GAL4-LB1, zones AMS2 and AMS3, do not overlap with that of the neurons labeled with LexAV-NV4. B–E: The neurons labeled with these

GAL4 lines innervate zone AMS1, the same area as that of the NV4-labeled neurons. Scale bar ¼ 10 lm in A1 (applies to A1–E4).



Figure 9. Projection patterns in the posterior maxillary sensory (PMS) branch. Single-channel images of the neurons labeled with each

GAL4 line (first columns; A1–H1) and superposition of the neurons labeled with the GAL4 lines (magenta) and NV4 (green) (second col-

umns; A2–H2). Same preparations as in Figure 8. Insets show the schematic diagrams of the projection patterns. (Dotted lines indicate

the projection areas of the NV4-labeled neurons.) Frontal view (dorsal up, lateral to the right). A,B: Neurons labeled with GAL4-LB4 and

LB5 terminate in the lateral subarea (zone PMS1) of the projection targets of the neurons labeled with LexAV-NV4. C: LB6-labeled neurons

arborize in the dorsal medial subarea (zone PMS2) as well as zone PMS1. D,E: LB2- and LB3-labeled neurons project to the ventral medial

subarea (zone PMS3) in addition to zones PMS1 and 2, i.e., all the areas contributed by the NV4-labeled neurons. F,G: Gr32a- and 66a-la-

beled neurons terminate across zones PMS1-3. H: Gr47a-labeled neurons arborize in the zones PMS2 and 3 but not in PMS1. Scale bar ¼10 lm in A1 (applies to A1–H2).

Miyazaki and Ito


We then examined the projection site of the GRNs that

detect sweetness. The sugar-responsive neurons labeled

with Gr5a-GFP (Fischler et al., 2007) matched exactly

with the projection sites of the GRNs labeled with GAL4-

NP1017 (Fig. 10C). Thus, Gr5a-expressing neurons also

arborize in zones PMS4 and 5.

Three zones are identified in the labellarsensory branch

LB1 and 2 labeled neurons that innervate the LS

branch (Fig. 11). Their axons formed two bundles before

they reached the target area. The major bundle bent pos-

teriorly at the root of the labial nerve and projected

through the ventralmost area of the SOG, whereas the

minor one shortly projected straight and branched off

from the ventral area of the AST to run posteriorly through

the more dorsal area of the SOG (Fig. 11A1,A2). The two

branches eventually merged to form a single target arbo-

rization. Whereas GAL4-LB2 and LexAV-NV4 labeled both

bundles (Fig. 11A), GAL4-LB1 labeled only the ventral

bundle (Fig. 11B).

Neurons labeled with LB2 terminated in the posterior

part of the LS branch, which overlapped with the area la-

beled with NV4. This projection site had a small protru-

sion on its ventral side, which was contributed by neurons

labeled with both LB2 and NV4 (arrows in Fig. 11A1,A2).

Because we noticed some neurons that arborized only in

the main part but not in the ventral protrusion (data not

shown), we named the former zone LS1 and the latter

zone LS2 (Fig. 11C,F). Axons labeled with LB1 terminated

in a segregated region that was anterior medial to the

projection sites labeled with NV4 and LB2. We therefore

named this area zone LS3 (Fig. 11B,F).

GAL4-LB1 also labeled some neural fibers projecting

via the cervical connective (arrowheads in Fig. 11B).

These fibers ran anteriorly from the cervical connective,

turned laterally, entered the LS branch of the PGC from

its lateral side, and terminated in the anterior medial area

of zone LS3. The cervical fibers labeled with LB1 were

thicker than the labeled fibers projecting from the labial

nerve.

Comparison with Gr66a-expressing neuronsSome or all the GRNs labeled with LB2–6 terminated

in zones PMS1–3, which overlapped with the target

areas of the GRNs expressing Gr32a, 47a, and 66a. Do

Figure 10. Projection areas of the water- and sugar-sensitive GRNs in the posterior maxillary sensory (PMS) branch, which do not overlap

with that of the NV4-labeled neurons. Single-channel images (A1,C1,C2) and superposition of the two signals (A2,C3). Frontal view (dorsal

up, lateral to the right). A: Most of the water-sensitive GRNs labeled with GAL4-NP1017 > UAS-DsRed arborize in the area that is more

ventral and lateral to the arborization areas of the NV4-labeled neurons (zone PMS4). In addition, a bundle of fibers projects to the central

medial area (zone PMS5). B: Schematic diagrams of the zones PMS4 and 5. (Dotted lines indicate the zones PMS1–3.) C: The sugar-

detecting neurons labeled with the Gr5a-GFP strain (C2, green in C3) also arborize in zones PMS4 and 5 but not in PMS1–3. Their projec-

tion targets overlap with those of the NP1017-labeled neurons (C1, magenta in C3). Scale bar ¼ 10 lm in A1 (applies to A1,2, C1–3).



Figure 12. Correlation of the projection patterns between newly identified cells and bitter-sensitive Gr66a-expressing neurons. A–G: Co-

localized signals in the labellum. High-magnification views of the cells labeled with each GAL4 line in the lateral sensilla of the labellum.

Single-channel images of the GAL4-expressing cells visualized with UAS-DsRed (first columns; A1–G1) and the cells expressing Gr66a-GFP

(second columns; A2–G2), and superposition images of both signals (third columns; A3–G3). LB3-labeled cells in the lateral sensilla and all

the cells labeled with LB6 express Gr66a (C,G). Expression does not overlap in LB1, LB2, and LB5 (A,B,D). LB4 labels both Gr66a-express-

ing (E) and Gr66a-non-expressing (F) cells. H–L: Projection areas in the PMS branch. Single-channel images of the GAL4-expressing cells

visualized with UAS-DsRed (first columns) and superposition images of the cells labeled with GAL4 and Gr66a-GFP (second columns). Fron-

tal view (dorsal up, lateral to the right). Neurons labeled with all the LB lines except for LB1 showed overlapping arborization with Gr66a-

expressing neurons in zones PMS1–3. Scale bar ¼ 10 lm in A1 (applies to A1–G3) and H1 (applies to H1–L2).

Miyazaki and Ito


map is more likely, we analyzed the axon terminals of

known pharyngeal and tarsal GRNs.

Some of the Gr genes are reported to be expressed in

the GRNs on the oesophagus (Thorne et al., 2004; Wang

et al., 2004). Among them, only the Gr2a gene labels

GRNs on the oesophagus but not in the labellum and legs

(Wang et al., 2004). We therefore utilized Gr2a-GAL4 for

specific visualization of the pharyngeal projection. To dis-

tinguish precise projection sites of the labeled axons, we

double-labeled the PGC with LexAV-NV4 (Fig. 14A,B). The

axons labeled with Gr2a-GAL4 projected via the pharyn-

geal nerve and entered the SOG at its anterior side. They

first ran into the anterior dorsal part of the SOG and

arborized in the area dorsal to AMS1. Because this brain

region was within the area called the ventral pharyngeal

sensory center (Ito et al., in preparation), we named this

projection target VPS1. The labeled axons further

extended medioposteriorly from VPS1 to the dorsolateral

edge of PMS1, and formed the second arborization area

in PMS1. Thus, the pharyngeal GRNs labeled with Gr2a-

GAL4 have one unique projection target (VPS1) as well as

the one shared with that of the labellar GRNs (PMS1).

Gr66a-GAL4 labels a subset of pharyngeal GRNs in

addition to the labellar GRNs (Thorne et al., 2004; Wang

et al., 2004). The pharyngeal fibers labeled with Gr66a-

GAL4 entered the SOG and formed two branches (Fig.

14C,D). One branch shared the same pathway as that of

the Gr2a-expressing GRNs, arborizing in VPS1 and

Figure 13. Distribution of the presynaptic sites. Superposition of the cytoplasmic signal of DsRed (magenta) and presynaptic sites-tar-

geted signals of n-syb::GFP (green), both of which are expressed by the GAL4 drivers. The projection areas with presynaptic sites appear

green to white. A,B: Reconstructed image of the anterior maxillary sensory (AMS) branch. First columns: frontal view (dorsal up, lateral to

the right), second columns: lateral view (dorsal up, anterior to the left). The labeled projection targets of zone AMS1 (A) and zones AMS 2

and 3 (B) have synaptic connections. C,D: Posterior maxillary sensory (PMS) branch. Orientation of the images is the same as in A and B.

The labeled projection targets of zones PMS1 and 2 (C) and zones PMS 4 and 5 (D) have synaptic connections. The axon bundles that

project to zones PMS1 and 2 have no synaptic connection in zone PMS4 (C). E: Labellar sensory (LS) branch. First panel: lateral view (dor-

sal up, anterior to the left), second panel: dorsal view (anterior down, lateral to the right). The labeled projection target of zone LS3 has

synaptic connections. Scale bar ¼ 10 lm in A1 (applies to A1–E2).



Figure 14

Miyazaki and Ito


projecting to the dorsolateral PMS1. The other branch ran

more ventrally and formed sparse arborization in the area

medial to AMS1 and dorsal to AMS3. We named this

arborization area VPS2. Fibers further extended posteri-

orly and entered the ventromedial part of PMS1. Thus,

the pharyngeal GRNs labeled with Gr66a-GAL4 have two

unique projection targets (VPS1 and VPS2) and one

shared with that of the labellar GRNs (PMS1). The pharyn-

geal nerve and zones VPS1 and 2 were also labeled

weakly with LexAV-NV4.

GRNs in the tarsi of the legs express some members of

the Gr gene family (Thorne et al., 2004; Wang et al.,

2004). Among them, the axons of the GRNs that express

Gr5a terminate in the thoracic ganglia, whereas the GRNs

that express Gr66a or 32a send their axons not only to

the thoracic ganglia but also to the posterior portion of

the SOG (Wang et al., 2004; Inoshita, 2007). It is likely

that Gr32a-expressing tarsal GRNs are subsets of those

that express Gr66a (Koganezawa et al., 2010). Single-cell

analysis of the Gr66a-expressing neurons showed that all

the neurons examined terminate in the SOG, regardless

of whether they have arborizations in the thoracic ganglia

(Inoshita, 2007). We therefore analyzed the projection

targets of the tarsal GRNs using double-labeling with

Gr66a-GAL4 and LexAV-NV4 (Fig. 14C,D).

As described earlier, Gr66a-expressing labellar and

pharyngeal GRNs terminate in the PMS branch. However,

we were able to trace the trajectory of the Gr66a-express-

ing tarsal GRNs, because they were spatially distinct from

that of the labellar and pharyngeal counterparts (Fig.

14E,F). Their axons entered the posterior SOG via the cer-

vical connective, ran along the anterior cerebrocervical

fascicle (aCCF), which passes between both sides of the

LS branches, and reached the area just posterior to zone

PMS1. We named this area of the aCCF terminals aCCF-

G1 (G for gustatory). The arborization areas of aCCF-G1

and PMS1 were adjacent to each other. Nevertheless, the

trajectory of the axon branches in aCCF-G1 did not

extend to PMS1 in eight of the nine cases we examined.

Thus, unlike Gr2a- and Gr66a-expressing pharyngeal

GRNs, Gr66a-expressing tarsal GRNs did not share pro-

jection targets with labellar GRNs.

The architecture of all the fiber bundles and terminals

contributed by the labellar, pharyngeal, and tarsal neu-

rons analyzed in this study are summarized in Figure

14G–I and Supplementary Movie S1.

DISCUSSIONS

After screening about 4,000 GAL4 enhancer-trap

strains, we obtained six lines that label various types of

GRNs and mechanosensory neurons in the labellum

rather preferentially. The labeled neurons sent their axons

to a wider variety of areas in the SOG than were reported

previously by visualizing the GRNs that express known Gr

genes. Whereas the projection targets of the neurons we

identified innervate in total nine zones of the PGC

(AMS1–3, PMS1–3, and LS1–3), GRNs with known Gr

genes (Thorne et al., 2004; Wang et al., 2004) innervate

only five zones (PMS1–5). In addition, water-sensing

GRNs labeled with GAL4-NP1017 and CO2-responsive

GRNs labeled with GAL4-E409 (Inoshita and Tanimura,

2006; Fischler et al., 2007) projected to zones PMS4, 5

and zone AMS1, respectively. Thus, five zones (AMS2, 3,

and LS1–3) were newly identified in this study. Among

Figure 14. Terminals of the GRNs of other sensory organs. A,B: Pharyngeal GRNs projecting via the pharyngeal nerve. Single-channel

images of the neurons labeled with Gr2a-GAL4 (A1,B1) and superposition of the neurons labeled with Gr2a-GAL4 (magenta) and LexAV-

NV4 (green) (A2,B2). Colored dashed lines indicate the projection areas of the pharyngeal neurons. Insets show the schematic diagrams

of the projection patterns, with dotted lines indicating the projection areas of the NV4-labeled neurons via the labial nerve. A: Frontal view

(dorsal up, lateral to the right). B: Lateral view (dorsal up, anterior to the left). Neurons expressing Gr2a-GAL4 project only via the pharyn-

geal nerve. The projection targets of these neurons are distributed in the ventral pharyngeal sensory center (zone VPS1) and the posterior

maxillary sensory center (zone PMS1). VPS1 lies laterally and anteriorly to PMS1, where the terminals of the pharyngeal projection over-

lapped with those of the labial projection. C–F: Terminals of the pharyngeal GRNs and putative tarsal GRNs projecting via the cervical con-

nective, labeled with Gr66a-GAL4. C,D: Single-channel images of the neurons (C1, D1) and superposition of the neurons labeled with

Gr66a-GAL4 (magenta) and NV4 (green) (C2, D2). E,F: Signals in the aCCF. Same preparations as in C and D, but the signals in the VPS

and PMS zones as well as in the antenno-suboesophageal tract (AST) are erased to show the projection targets of the tarsal GRNs clearly.

C,E: Frontal view (dorsal up, lateral to the right), D,F: Lateral view (dorsal up, anterior to the left). The axons of the pharyngeal GRNs arbo-

rize in two areas (zones VPS1 and 2) before entering PMS1. Tarsal GRNs project through the anterior cerebrocervical fascicle (aCCF) to

terminate in the region posterior to zone PMS1 (aCCF-G1). Asterisks and arrowheads indicate the entry points of the labial nerve (LbN)

and pharyngeal/accessory pharyngeal nerve (PhN), respectively. Insets show the schematic diagrams of the projection patterns. (Dotted

lines indicate the projection areas of the NV4-labeled neurons via the labial nerve.) G–I: Schematic model of the pharyngeal and tarsal pro-

jections and the 11 zones of the PGC that receive labial projections. Anterior (G), dorsal (H), and posterior lateral oblique views (I). AMS,

anterior maxillary sensory; OES, oesophagus. A movie of the rotating model is available as Supplementary Movie S1. Scale bar ¼ 10 lmin A1 (applies to A1–B2) and C1 (applies to C1– F2).



them, three zones (AMS2, 3 and LS3) are contributed to

specifically by putative mechanosensory neurons labeled

with LB1, and two zones (LS1, 2) receive terminals from

putative GRNs. None of the known GRNs innervate these

LS zones.

In contrast, our set of GAL4 driver lines did not visual-

ize GRNs that project to zones PMS4 and 5, suggesting

that our screening was not comprehensive enough. Dur-

ing the course of screening, we discarded tens of GAL4

lines because they labeled not only GRNs but also many

ORNs in the maxillary palp or other sensory neurons in

the head capsule. Some of these lines may label GRNs

that innervate PMS4 or 5. Indeed, NP1017 was not

selected in our screening because it shows strong expres-

sion in the maxillary palp.

Previous studies suffered from low resolution for the

identification of the projection targets of the GRNs. To

overcome this problem, we utilized a newly established

LexAV enhancer-trap strain to reproducibly label defined

sets of GRNs independent of GAL4 drivers. This allowed

examination of fine subareas within the PGC, leading to

the unambiguous identification of 11 zones. This study

thus proved the advantage of combining two different

types of enhancer-trap systems for the precise mapping

of brain neurons.

Projection patterns identified in earlierstudies

Before genetic labeling of specific GRNs became avail-

able, several studies were performed to trace the projec-

tions from the labellum to the brain of the flies. Stocker

and Schorderet (1981) reported that the labellar GRNs

send their axons to three regions that are aligned along

the anterior-posterior axis in the SOG, which seem to cor-

respond with the three PGC branches we described in

this study. The neurons connected to the bristles on the

lateral surface of the labellum are reported to project

specifically to the posterior two regions. In our study,

most of the GRNs in the lateral sensilla projected to the

PMS branch; only GRNs in the medial taste pegs had

arborization in the LS branch (Table 1). It has not yet

been determined whether the posterior branch reported

by Stocker and Schorderet (1981) might correspond to a

part of the PMS branch or represent the terminals of yet

unidentified types of GRNs.

Based on single-cell projection analysis using Golgi

impregnation, Nayak and Singh (1985) classified seven

types of neurons that enter the SOG via the labial nerve.

Among them, type I and type II neurons have large arbori-

zation areas in the SOG, which roughly correspond to the

entire AMS, PMS, and LS branches we identified. Type

III–VII neurons, on the contrary, project to smaller, more

specific areas of the PGC. Injection of horseradish peroxi-

dase (HRP) to the neurons in the lateral sensilla visualized

only type I, II, and IV–VII neurons (Shanbhag and Singh,

1992), suggesting that type III neurons derive from the

medial taste pegs. The arborization area of the type III

neurons appears to correspond to zone AMS1. Type IV

and VII neurons, which send axons to the dorsal and ven-

tral regions of the ipsilateral SOG, are likely to project to

zones PMS1 and PMS4, respectively. Type VI neurons ter-

minate in the medial area, which is slightly dorsal to the

terminals of the type V neurons. The projection areas of

type V and VI neurons may therefore correspond to zones

PMS3 and PMS2, respectively, or, alternatively, the for-

mer may arborize in PMS5 whereas the latter terminate in

both zones PMS2 and 3.

Neurons labeled with molecular-genetic techniques

corresponded well to these classified projection patterns.

The projection area of the GRNs in the medial taste pegs

labeled with E409 and NP1017 matches that of the type

III neurons, which is the only cell type that was not la-

beled by HRP injection into the lateral sensilla (Shanbhag

and Singh, 1992). The GRNs labeled with LB4/LB5 and

Gr5a match that of type IV and type VII, respectively. The

GRNs expressing Gr47a seem to correspond to type V or

VI neurons. Specific projections of type III–VII neurons do

not cover zones AMS2, 3 and LS1–3. Only wide-field pro-

jections of type I and type II neurons cover these zones.

Some of the neuron types identified in this study, such as

those labeled in LB1 and LB2, may arborize in multiple

branches as type I and II neurons do.

Ipsilateral projections like zone PMS1 and contralat-

eral projections like zones PMS2 and 3 of the putative

gustatory neurons were also reported in the blowfly

Phormia regina (Yetman and Pollack, 1986; Edgecomb

and Murdock, 1992). Moreover, putative mechanosen-

sory neurons in the blowfly labellum project to a more

posterior area in the SOG (Edgecomb and Murdock,

1992), which seems to correspond to zone LS3. These

studies did not describe the projections that should cor-

respond to the AMS branch we found in the fruitfly SOG.

Whether this is due to the limited resolution of the previ-

ous studies along the anterior-posterior axis or is a

reflection of the differences between species remains to

be resolved.

Recent studies that visualized GRNs by using molecu-

lar-genetic tools revealed only parts of the projection pat-

terns that have been described in studies using classic

techniques. Projections to only AMS and PMS branches

had been reported, and the PMS branch had been classi-

fied into only two regions. By labeling a wider variety of

GRNs and taste-associated mechanosensory neurons, we

were able to identify most of the projection targets that

have classically been reported.

Miyazaki and Ito


Correlation between the locations of cellbodies in the labellum and projectiontargets in the PGC

In the labellum of Drosophila, taste receptors reside in

the lateral sensilla and medial taste pegs (Falk et al.,

1976; Nayak and Singh, 1983). Do GRNs in these two

areas innervate different zones of the PGC? LB2, which

labels about 20 neurons in the medial taste pegs, visual-

ized axons that project to all the three branches. In con-

trast, all the GRNs labeled with E409 (Fischler et al.,

2007) reside in the medial taste pegs and project only to

zone AMS1. Although NP1017 labels neurons in both the

lateral sensilla and medial taste pegs, only the latter

houses the cells that project to the AMS branch (Inoshita

and Tanimura, 2006). Considering that both E409 and

LB2 label more than two-thirds of the GRNs in the medial

taste pegs, it is rather likely that a large portion of the

LB2-labeled cells coincide with the cells that are labeled

with E409 and/or NP1017 and project only to zone

AMS1. The remaining few cells should comprise a novel

cell type, which projects to the PMS and LS branches.

Indeed, LB2 labeled projections to the PMS and LS

branches more faintly than those to the AMS branch, sug-

gesting that a smaller number of neurons contributes to

these branches.

As discussed earlier, Golgi impregnation analysis iden-

tified no neurons that arborize specifically in the area that

corresponds to the LS branch, which is contributed to

only by the wide-field type I and type II neurons (Nayak

and Singh, 1985). It is therefore plausible that the GRNs

labeled with LB2 but not with E409 and NP1017 may con-

tain wide-field neurons that arborize in all three branches.

Future single-cell projection analysis of the LB2-labeled

neurons would reveal how gustatory signals from single

taste-peg GRNs would be transmitted to the PMS and LS

branches in the PGC.

Among the GRNs identified in previous studies, cells

that express known Gr genes are located only in the lat-

eral sensilla of the labellum and send their axons only to

the PMS branch (Thorne et al., 2004; Wang et al., 2004).

Water-detecting neurons located in the lateral sensilla

also project to the PMS branch (Inoshita and Tanimura,

2006). Consistent with these data, most of the GRNs we

identified in the lateral sensilla, labeled with LB4–6, inner-

vate only this branch (Table 1).

LB3 labels neurons in both lateral sensilla and medial

taste pegs. Considering the preferential projection pat-

terns of the GRNs from the medial and lateral parts of the

labellum to the AMS and PMS branches of the PGC,

respectively, it is likely, but not yet confirmed, that the

LB3-labeled projections in zone AMS1 would derive from

the medial taste pegs and those in zones PMS1–3 from

the lateral sensilla.

Our study identified two types of GRNs in the lateral

part of the labellum. Whereas LB3–5 labeled relatively

small neurons, LB6 labeled neurons with large cell bodies.

The terminals of the small and large neurons largely over-

lap in the PMS branch, suggesting that the two types of

neurons do not send information to segregated areas of

the PGC.

Drosophila larvae have four gustatory sensory organs on

the head (Stocker, 1994). GRNs from each of these organs

innervate different areas of the brain, forming a kind of

topographic map in the larval brain (Colomb et al., 2007).

Most GRNs in the adult lateral sensilla project to the PMS

branch, whereas the CO2-responsive GRNs in the medial

taste pegs innervate the AMS branch. Although these

appear to suggest similar spatial segregation of sensory

organs and target neuropils both in larvae and adults, we

found that some of the LB2-labeled GRNs in the medial

taste pegs project in an overlapping fashion to the PMS

branch. This finding suggests that at least some kind of

spatial conversion would occur between the signals arising

from the lateral and medial parts of the labellum.

Estimated number of the identified cellsThe Drosophila labellum reportedly has about 30 gusta-

tory sensilla and 2 mechanosensory sensilla on its lateral

part, and about 30 taste pegs on its medial recess. The

numbers of the sensilla and taste pegs seem to vary

somewhat (Stocker 1994; Singh, 1997; Shanbhag et al.,

2001; our observations). Each taste peg has one GRN

and one mechanosensory neuron, whereas each gusta-

tory sensillum houses either two or four GRNs and one

mechanosensory neuron. The number of total GRNs asso-

ciated with these sensilla is estimated to be about 100

(Stocker 1994; Singh, 1997). Thus, there should be in

total about 130 GRNs and 60 mechanosensory cells

associated with the sensilla and pegs.

It is likely that each of the ca. 30 lateral sensilla is

associated with one bitter-detecting neuron expressing

Gr66a and one sugar-responding neuron expressing Gr5a

(Hiroi et al., 2002; Thorne et al., 2004; Wang et al., 2004).

Therefore, there should be about 60 neurons of these

kinds. Among the GRNs identified previously, those that

express Gr22b, 22e, 22f, 28be, 32a, 47a, 59a, and 59b,

and perhaps 28ba and 28bd, are subsets of the Gr66a-

expressing neurons (Thorne et al., 2004; Wang et al.,

2004; Thorne et al., 2008), those expressing Gr61a and

64f are overlapping population or subsets of the Gr5a-

positive neurons (Dahanukar et al., 2007), and Gr28a and

28bc are likely to be expressed in subsets of the cells

that express either Gr5a or 66a (Thorne et al., 2008).

Therefore, they all seem to be included in the number of

the cells that express Gr5a or 66a. Many cells that

express Gr33a, 39ad, 59f, 64b, 64c, and 64e also co-



express Gr5a or 66a (Jiao et al., 2007), but it is not known

whether all of them fall into this criterion. In addition,

about 20 of the 30 lateral sensilla are associated with

water-detecting neurons that are labeled with NP1017

(Hiroi et al., 2002; Inoshita and Tanimura, 2006). Thus, at

least 80 GRNs had already been identified.

In this study, we identified various types of GRNs. The

number of the identified cells shown in Table 1 might be

slightly underestimated, because the UAS-DsRed reporter

used for the cell count tended to label slightly smaller num-

bers of cells than those visualized with the UAS-GFP re-

porter due to the weaker expression level (our unpublished

observation). Among the sensilla-associated GRNs identi-

fied in this study, about 3 of the 17–18 cells labeled with

LB4 and about four and three cells labeled with LB3 and

LB6, respectively, express Gr66a. They are therefore sub-

sets of the known bitter-detecting GRNs. The remaining

about 14 GRNs labeled with LB4 and the two neurons la-

beled with LB5 do not express Gr66a and innervate neither

AMS1 nor PMS4/5, the targets of the known water- and

sugar-detecting neurons. Thus, they should represent

novel cell types. Although it is not known whether the cells

labeled with LB4 and LB5 overlap, the total count of these

cells would be 14–16, i.e., about 15 cells. All together,

about 95 of the estimated 100 sensilla-associated neurons

have been described. It is therefore plausible that only a

handful GRNs remain to be identified in the lateral sensilla.

In the medial recess, the GRNs labeled with NP1017

and E409 overlap (Fischler et al., 2007). LB3 labels several

GRNs in the medial taste pegs and projections to zone

AMS1. LB4 sometimes labels neurons in the taste pegs,

and projections to the AMS branch were also observed in

some cases (data not shown). Considering that E409 labels

a large part of the taste peg-associated GRNs, it is likely

that the taste-peg neurons labeled with LB3 and LB4 are

subsets of the E409-positive cells. As discussed in the pre-

vious section, some of the neurons labeled with LB2 should

also overlap the E409-labeled neurons. The remaining sev-

eral LB2-positive cells are likely to be the only novel cell

type identified in the medial recess. Considering that both

E409 and LB2 label about two-thirds of the ca. 30 taste-

peg GRNs, it is likely that most of the taste peg-associated

GRNs have already been identified.

The LB1 strain labels about 20 mechanosensory cells

in the medial recess. The remaining neurons in the medial

recess (ca. 10 cells) and all the mechanosensory neurons

associated with the lateral chemosensilla (ca. 30 cells)

are yet to be identified.

Functions of the labeled GRNsElectrophysiological analyses revealed that certain

GRNs can be activated by bitter compounds such as caf-

feine, quinine, berberine, denatonium, etc. (Meunier

et al., 2003; Hiroi et al., 2004). Flies indeed exhibit avoid-

ance behaviors against these compounds through the

mediation of the GRNs that express the Gr66a gene

(Thorne et al., 2004; Wang et al., 2004). However, Gr66a

functions as a receptor only for caffeine with a co-recep-

tor, Gr93a (Moon et al., 2006; Lee et al., 2009). The fact

that several other Gr genes such as Gr28a, 32a, and 47a

are expressed within the subsets of Gr66a-positive neu-

rons raises the possibility that these neural subsets may

be equipped for detecting specific substances within the

variety of bitter compounds (Thorne et al., 2004; Wang

et al., 2004). We showed that these neurons send their

axons to specific subareas of the projection targets inner-

vated by the Gr66a-expressing neurons. For example, the

neurons that express Gr47a project only to zones PMS2

and 3. Similarly, GAL4-LB6 labeled a subset of Gr66a-

expressing neurons that send their axons only to zones

PMS1 and 2. These findings suggest that the three zones

of the PMS branch are likely to process different combi-

nations of bitterness-associated stimuli. Further experi-

ments with behavioral (Thorne et al., 2004; Wang et al.,

2004; Inoshita and Tanimura, 2006; Fischler et al., 2007)

and physiological (Marella et al., 2006; Fischler et al.,

2007; Hiroi et al., 2008) approaches using genetically

modified animals would resolve the validity of this

hypothesis.

The expression patterns of GAL4 enhancer-trap strains

are likely to mimic, at least to some extent, the expres-

sion patterns of certain genes near the GAL4 insertion

sites. Because LB1–5 strains showed expression not only

in the GRNs but also in some ORNs, mechanosensory

neurons, and non-neuronal cells, the genes associated

with the observed expression pattern may not be related

exclusively to taste detection. In contrast, the LB6 strain

showed a rather specific expression pattern in the Gr66a-

expressing GRNs, suggesting that the associated gene

may play important roles in bitter-taste detection. The

genes near the insertion site of each GAL4 strain are

summarized in Table 3. No known Gr genes or molecules

that may function as taste receptors were found, suggest-

ing that the genes associated with the GAL4 lines identi-

fied may function indirectly in the development, signal

transduction, or maintenance of the labeled GRNs.

Integration of mechanosensory andgustatory information in the PGC

LB1 is unique in that it is likely to label not GRNs but

mechanosensory neurons in the medial taste pegs. A

taste peg is associated with one chemosensory neuron

and one mechanosensory neuron (Nayak and Singh,

1983). The CO2-responsive neurons labeled with GAL4-

Miyazaki and Ito


NP1017 and E409 also lie in the medial taste pegs (Inosh-

ita and Tanimura, 2006; Fischler et al., 2007). The chemo-

and mechanosensory signals from the taste pegs, how-

ever, appear to be conveyed to different zones of the

PGC. The GRNs sensitive for CO2 send their axons to

zone AMS1, whereas mechanosensory neurons labeled

with LB1 project to zones AMS2, 3, and LS3.

Similar separated projections of gustatory and tactile

neurons have been reported in various insect species. As

mentioned above, the presumptive mechanosensory neu-

rons in Phormia project to the more posterior region in

the SOG than the gustatory neurons do (Edgecomb and

Murdock, 1992). In the fruitfly and blowfly, as well as in

crickets and locusts, chemo- and mechanosensory neu-

rons on the leg sensilla send their axons to distinct

regions of the thoracic ganglia (Johnson and Murphey,

1985; Murphey et al., 1989; Newland, 1991). In the cock-

roach Periplaneta americana, contact chemosensory and

tactual neurons on the same sensillum on the antenna in-

nervate segregated areas in the brain (Nishino et al.,

2005). In contrast, leg neurons of a locust, Schistocerca

gregaria, exhibit indistinguishable projection betweens

chemosensory and mechanosensory modalities (Newland

et al., 2000).

The reason why all the gustatory sensilla are associ-

ated with one mechanosensory neuron is yet unknown,

but considering that information from GRNs should be

regarded as noise if the sensilla are not in contact with

objects, mechanoreceptors are likely to detect subtle dis-

placement of the sensilla caused by the contact. To asso-

ciate this contact signal with the signals from GRNs of

the same sensilla, the brain must integrate information

from these two types of neurons. Nevertheless, the termi-

nals of the putative mechanosensory neurons labeled

TABLE 3.

Genes Near the Insertion Sites of the Identified GAL4 Enhancer-Trap Strains1

Strain Locus

Insertion

direction

Nearby

gene

Direction

of gene

Distance

(kb) Annotation/predicted domain

LB1 - (3) � CG40182 � �139 Histone 2A

CG40188 � 5 —CG40189 þ 40 —CG40191 þ 107 —

LB2 25E3 � CG7277 � �41 Monooxygenase, UbiH, 2-polyprenyl-6-methoxyphenol hydroxylase and related

FAD-dependent oxidoreductases

CG7382 � �30 —CG14020 þ �28 —CG6634 þ 22 T-box DNA binding domain of the T-box

family of transcriptional regulators

CG31647 � 41 —H15 þ 74 T-box DNA binding domain of the T-box

family of transcriptional regulators

LB3 � (X) — — — — —LB4 68F1 þ CG6928 þ �50 Sulfate transporter

CG6793 þ �13 —rols � 0 rolling pebbles ankyrin repeats,

Tetratricopeptide repeat domain

Sema-5c � 10 Semaphorin domain

CG17154 þ 31 —CG6801 þ 60 —

LB5 � (3) — — — — —LB6 17C1 þ CG6394 � �10 UDP-N-acetyl-a-D-galactosamine:polypeptide

N-acetylgalactosaminyltransferase 2Rad51D þ �9 DNA recombinase

Wnt5 � �4 Wnt oncogene analog 5CG6461 � 0 c-GlutamyltranspeptidaseCG6470 � 4 —CG6335 þ 6 Histidyl-tRNA synthetase

CG6481 � 13 —

1Information about the insertion sites and neighboring genes was obtained from the GETDB database (Hayashi et al., 2002). The loci of the inser-

tion sites and directions of the inserted GAL4 are shown in the second and third columns. The fourth through sixth columns show the names, direc-

tions, and distance from the GAL4 insertion sites (þ/� according to the base pair number of the genome data) of up to three nearby genes in the

upstream and downstream regions of the genome. The rightmost column shows the annotation reported in GETDB (in roman letters) or containing

domains predicted by NCBI Conserved Domain Search (in italic letters). In LB6, the GAL4 transgene is inserted within the CG6461 gene. Insertion

of LB1 is found in a genomic DNA fragment that contains several nearby candidate genes but whose location in the genome has not been identi-

fied. Precise insertion sites are not identified for LB3 and LB5. (X) and (3) indicate the chromosomes that should contain the insertion.



with LB1 do not overlap any of the GRNs. Mechanosen-

sory information is likely to be transmitted to zones

AMS2, 3 and LS3 of the PGC, whereas gustatory informa-

tion is sent to zones AMS1, PMS1–5, and LS1, 2. Integra-

tion between mechanosensory and gustatory signals,

therefore, does not seem to occur at the initial level of

the PGC.

Interestingly, the PMS branch, in which sugar- and bit-

terness-responding GRNs terminate, is not associated

with the zones with mechanosensory terminals. Because

LB1 labels only neurons in the medial taste pegs and two

mechanosensilla on the lateral surface, the projection tar-

gets of about 30 mechanosensory neurons in the lateral

gustatory sensilla are yet unknown. Screening further

strains for visualizing such neurons would be important to

figure out whether mechanosensory information from

these sensilla is also sent to zones AMS2, 3 and LS3.

Integration of multiple types of gustatoryinformation in the PGC

Previous studies revealed that GRNs with different

taste modalities transmit signals to different areas of the

PGC. The sugar-responsive Gr5a-expressing neurons pro-

ject their axons to zones PMS4 and 5, whereas bitter-

detecting Gr66a-positive neurons innervate zones PMS1–

3 (Thorne et al., 2004; Wang et al., 2004), and CO2-

detecting neurons labeled with E409 arborize in zone

AMS1 (Fischler et al., 2007). Our study, however, identi-

fied a few cases in which neurons responsible for differ-

ent gustatory modalities may converge to the same zone

of the PGC. LB4 and LB5 label the neurons that do not

express Gr66a and therefore may not detect bitterness.

Even so, neurons labeled with these lines feed into zone

PMS1, a part of the area responsible for bitter taste. The

gustatory modality detected by these neurons is as yet

unknown. They may detect other kinds of taste, such as

salt or sour, or detect certain other types of bitter

compounds.

A clearer conversion of different gustatory modalities

occurs between sweetness and the sense of water. Inosh-

ita and Tanimura (2006) reported that the GRNs labeled

with NP1017 are responsible for the detection of water

and that their projection target in the SOG was distinct

from that of the Gr66a-expressing GRNs. We found that

the water-detecting neurons terminate in zones PMS4

and 5, which spatially overlap with the areas innervated

by the sweet-detecting GRNs that express Gr5a.

Thus, the PGC areas that are responsible for bitter and

sweet sensation both receive some other modalities of

taste information. Unlike integration between mechano-

sensory and gustatory information, integration between

certain combinations of different gustatory sensations is

therefore likely to occur at the level of the primary center.

Partial topographic specificity betweenlabellar and other projections

In addition to the projection targets of the GRNs that

reside in the labellum, in this study we also analyzed the

innervation patterns of the pharyngeal and tarsal GRNs in

the brain by labeling the neurons that express Gr2a and

66a genes. As mentioned earlier, GRNs deriving from the

four sensory organs in the Drosophila larva project to dis-

tinct areas of the brain (Colomb et al., 2007). The axons

of the tarsal GRNs labeled with Gr66a-GAL4 appear to ter-

minate in the area distinct from the terminals of the label-

lar GRNs, showing similar sense organ-dependent topo-

graphical separation. In contrast, the pharyngeal GRNs

visualized with Gr2a-GAL4 and Gr66a-GAL4 terminate in

the area that partially overlaps the terminals of the label-

lar GRNs, indicating a certain level of sensory integration

from the two sense organs associated with the mouth.

Because we were not able to analyze the targets of all the

pharyngeal and tarsal GRNs systematically, it is possible

that other GRNs may have different projection patterns.

The projection targets of the GRNs on the oesopha-

gus have at least three zones. Two are pharyngeal

specific (VPS1, 2) and the other is shared by the

labellar projections (PMS1). Zone PMS1 is associated

primarily with bitter sensation, and Gr66a-expressing

neurons of not only the labellum but also the oeso-

phagus terminate in PMS1. In contrast, Zone PMS1 is

also contributed by the labellar GRNs labeled with LB4

and LB5, which do not express Gr66a. Because the

function of the Gr2a-expressing pharyngeal GRNs

remains unknown, it is yet to be determined whether

they are involved in bitter sensation. Zone PMS1 is

therefore a site of convergence between heterogene-

ous signals deriving from Gr66a-positive and -negative

labellar GRNs as well as subsets of pharyngeal GRNs.

The specific and shared arborization areas of the pha-

ryngeal GRNs may imply their different roles in gustatory

perception. For example, the overlapping region (PMS1)

may integrate gustatory stimuli from the labellar and pha-

ryngeal sensory organs, whereas the activity in the pha-

ryngeal-specific region (VPS1 and 2) may allow discrimi-

nation of the stimulated site. Compared with the tightly

bound terminals of the labellar GRNs in the AMS and

PMS branches, arborizations of the pharyngeal GRNs in

VPS1 and 2 were much sparser, suggesting the existence

of many other neurons, intermingled with the pharyngeal

GRN terminals.

The axons of the tarsal GRNs labeled with Gr66a-

GAL4 terminate in part of the aCCF (zone aCCF-G1),

Miyazaki and Ito


which lies just posterior to zone PMS1. Thus, the

GRNs that express the same Gr66a gene terminate in

distinct brain regions depending on the sense organs

from which they derive, suggesting a topographical

rather than functional specificity in the gustatory sen-

sory map in this case.

Figure 15. Neurons other than the GRNs that are labeled in the LB and NV strains. Same preparations as shown in Figure 6, without era-

sure of irrelevant signals. Red-cyan 3D stereograms are available as Supplementary Figure S4. The erased signals are as follows. A: No

signals were erased for LB1. B: LB2; projections from the antennal mechanosensory and motor center (AMMC)/(arrow) and fibers from

the cervical connective (arrowheads). C: LB3; cell bodies of various neurons (arrows), which do not project to the PGC, and fibers from

the cervical connective (arrowheads). D: LB4; various neural fibers in the SOG. E: LB5; cells around the root of the pharyngeal nerve

(arrow). F: LB6; weakly labeled fibers from the cervical connective (arrowheads). G: NV4; projections from the AMMC (arrow) and fibers

from the cervical connective (arrowheads). AMS, anterior maxillary sensory; LS, labellar sensory; PMS, posterior maxillary sensory. Scale

bar ¼ 50 lm in A1 (applies to A1–G2).



Gr32a-GAL4 labels a subset of the Gr66a-expressing

GRNs in the legs (Koganezawa et al., 2010). They are

therefore likely to terminate also in zone aCCF-G1.

Although male flies court females actively, they seldom

attempt to court males and mated females. The

Gr32a-expressing GRNs in the forelegs mediate inhibi-

tory signals to this courtship behavior (Miyamoto and

Amrein, 2008). These GRNs are also required for a

step in the courtship ritual, the unilateral wing vibra-

tion (Koganezawa et al., 2010). The sexually dimorphic

interneurons that are also involved in the regulation of

the wing extension have synaptic connections to the

Gr32a-positive tarsal GRNs in the male SOG (Kogane-

zawa et al., 2010). Thus, it is plausible that zone

aCCF-G1 might be involved in processing the chemical

stimuli that are necessary for shaping male courtship.

In contrast, the tarsal GRNs that express Gr66a have

also been shown to be involved in avoidance to bitter sub-

stances (Wang et al., 2004). The GRNs that express

Gr66a but not Gr32a might be responsible for this. In this

regard it is important to note that aCCF-G1 is juxtaposed

to the terminals of the bitter-detecting GRNs in the label-

lum (PMS1–3). Secondary neurons that extend dendrites

to these neighboring regions might be involved in the

processing of the labellum- and leg-derived bitter stimuli.

Higher order gustatory neuronsWe confirmed that the terminals of the labeled GRNs

possess presynaptic sites, which should transmit gusta-

tory information to the secondary neurons that arborize in

the three PGC branches. Previous studies identified a few

candidate higher order gustatory neurons that have proc-

esses in the SOG (Melcher and Pankratz, 2005; Thorne

and Amrein, 2008). Limited knowledge about the detailed

projection map of the GRNs, however, made it difficult to

identify precisely the neurons that certainly arborize in

the PGC. A recent study reported a type of motor neuron

that is involved in the proboscis extension reflex invoked

by the attractive gustatory stimuli (Gordon and Scott,

2009). These neurons were shown to have few direct con-

nections with Gr5a-expressing GRNs, indicating that at

least one step of interneurons intervenes between the

GRNs and motor neurons. Appetitive and aversive taste

serve as unconditioned stimuli of reward and punishment,

respectively, for associative learning (Schwaerzel et al.,

2003; Unoki et al., 2005, 2006). The mushroom bodies

and octopaminergic and dopaminergic neurons are

reported to be essential for such learning (Schwaerzel

et al., 2003). The information pathway between the PGC

and these neurons is as yet unknown.

Our study not only provided detailed information about

the structure of the PGC but also significantly expanded

the areas of the SOG that are known to be innervated by

the putative GRNs and taste-associated mechanosensory

neurons. This should increase the variety of higher order

neurons that may receive gustatory information. More-

over, we identified a convenient landmark strain, LexAV-

NV4, which labels 6 of the 11 zones—and 6 of the 8 zones

that are contributed by the GRNs—in all three PGC

branches. This allows direct and detailed assessment of

the GAL4-expressing neurons that would potentially arbo-

rize in the PGC. Studies for identifying second-order gus-

tatory neurons based on the systematic structural infor-

mation about the PGC provided by this study will uncover

the neural processes connecting the taste information

input and taste-invoked behavior, or gustatory stimuli and

taste-conditioned memory.

ACKNOWLEDGMENT

We thank J. Urban and G. Technau for MZ series

enhancer-trap strains, the members of the NP consortium

and D. Yamamoto for the NP series strains, S. Takeuchi,

K. Endo, T. Awasaki, and T. Lee for LexAV enhancer-trap

strains, K. Scott for Gr-GAL4 and Gr-GFP transgenic

strains and the GAL4-E409 strain, L. Vosshall for Or83b-

GAL4 transgenic flies, E. Buchner and A. Hofbauer for the

nc82 antibody, the Developmental Studies Hybridoma

Bank for anti-ELAV and anti-Synapsin antibodies, and B.

Dickson and the Bloomington Stock Center for UAS-re-

porter strains. We thank S. Shuto, M. Matsukuma, and K.

Yamashita for technical assistance and K. Shinomiya and

K. Endo for helpful discussions and sharing unpublished

data.

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neural architecture of the primary gustatory center of ... · phageal ganglion (sog) of the brain....

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